Plant cellulose synthase and promoter sequences

ABSTRACT

Provided are two plant cDNA clones that are homologs of the bacterial CelA genes that encode the catalytic subunit of cellulose synthase, derived from cotton ( Gossypium hirsutum ). Also provided are genomic promoter regions to these encoding regions to cellulose synthase. Methods for using cellulose synthase in cotton fiber and wood quality modification are also provided.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 08/960,048filed Oct. 29, 1997, now U.S. Pat. No. 6,271,443 which claims priorityto U.S. application Ser. No. 60/029,987 filed Oct. 29, 1996.

TECHNICAL FIELD

This invention relates to plant cellulose synthase cDNA encodingsequences, and their use in modifying plant phenotypes. Methods areprovided whereby the sequences can be used to control or limit theexpression of endogenous cellulose synthase.

This invention also relates to methods of using in vitro constructed DNAtranscription or expression, cassettes capable of directing fiber-tissuetranscription of a DNA sequence of interest in plants to produce fibercells having an altered phenotype, and to methods of providing for ormodifying various characteristics of cotton fiber. The invention isexemplified by methods of using cotton fiber promoters for altering thephenotype of cotton fiber, and cotton fibers produced by the method.

BACKGROUND

In spite of much effort, no one has succeeded in isolating andcharacterizing the enzyme(s) responsible for synthesis of the major cellwall polymer of plants, cellulose.

Numerous efforts have been directed toward the study of synthesis ofcellulose (1,4-β-D-glucan) in higher plants. However, hampered by lowrates of activity in vitro, the cellulose synthase of plants hasresisted purification and detailed characterization (for reviews, see1,2). Aided by the discovery of cyclic-di-GMP as a specific activator,the cellulose synthase of the bacterium Acetobacter xylinum can beeasily assayed in vitro, has been purified to homogeneity, and acatalytic subunit identified (for reviews, see 2,3). Furthermore, anoperon of four genes involved in cellulose synthesis in A. xylinum hasbeen cloned (4-7).

Characterization of these genes indicates that the first gene, termedeither BcsA (7) or AcsAB (6) codes for the 83 kD subunit of thecellulose synthase that binds the substrate UDP-glc and presumablycatalyzes the polymerization of glucose residues to 1,4-β-D-glucan (8).The second gene (B) of the operon is believed to function as aregulatory subunit binding cyclic-di-GMP (9) while recent evidencesuggests that the C and D genes may code for proteins that form a poreallowing secretion of the polymer and control the pattern ofcrystallization of the resulting microfibrils (6).

Recent studies with another gram-negative bacterium, Agrobacteriumtumefaciens, have also led to cloning of genes involved in cellulosesynthesis (10,11), although the proposed pathway of synthesis differs insome respects from that of A. xylinum. In A. tumefaciens, a CelA geneshowing significant homology to the BcsA/AcsAB gene of A. xylinum, isproposed to transfer glc from UDP-glc to a lipid acceptor; other geneproducts may then build up a lipid oligosaccharide that is finallypolymerized to cellulose by the action of an endo-glucanase functioningin a synthetic mode. In addition, homologs of the CelA, B, and C geneshave been identified in E. coli, but, as this organism is not known tosynthesize cellulose in vivo, the function of these genes is not clear(2).

These successes in bacterial systems opened the possibility thathomologs of the bacterial genes might be identified in higher plants.However, experments in a number of laboratories utilizing the A. xylinumgenes as probes for screening plant cDNA libraries have failed toidentify similar plant genes. Such lack of success suggests that, ifplants do contain homologs of the bacterial genes, their overallsequence homology is not very high. Recent studies analyzing theconserved motifs common to glycosyltransferases using either UDP-glc orUDP-GlcNAc as substrate suggest that there are specific conservedregions that might be expected to be found in any plant homolog of thecatalytic subunit (referred to hereafter as CelA). In one of thesestudies, Delmer and Amor (2) identifed a motif common to many suchglycosyltransferases including the bacterial CelA proteins. Anindependent analysis (6) also concluded that this motif was highlyconserved in a group of similar glycosyltransferases.

Extending these studies further, Saxena et al. (12) presented an elegantmodel for the mechanism of catalysis for enzymes such as cellulosesynthase that have the unique problem of synthesizing consecutiveresidues that are rotated approximately rotated 180° with respect toeach other. The model invokes independent UDP-glc binding sites and,based upon hydrophobic cluster analysis of these enzymes, the authorsconcluded that 3 critical regions in all such processiveglycosyltransferases each contain a conserved aspartate (D) residue,while a fourth region contained a conserved QXXRW motif. The first Dresidue resides in the motif as previously analyzed (2,6).

In general, genetic engineering techniques have been directed tomodifying the phenotype of individual prokaryotic and eukaryotic cells,especially in culture. Plant cells have proven more intransigent thanother eukaryotic cells, due not only to a lack of suitable vectorsystems but also as a result of the different goals involved. For manyapplications, it is desirable to be able to control gene expression at aparticular stage in the growth of a plant or in a particular plant part.For this purpose, regulatory sequences are required which afford thedesired initiation of transcription in the appropriate cell types and/orat the appropriate time in the plant's development without havingserious detrimental effects on plant development and productivity. It istherefore of interest to be able to isolate sequences which can be usedto provide the desired regulation of transcription in a plant cellduring the growing cycle of the host plant.

One aspect of this interest is the ability to change the phenotype ofparticular cell types, such as differentiated epidermal cells thatoriginate in fiber tissue, i.e. cotton fiber cells, so as to provide foraltered or improved aspects of the mature cell type. Cotton is a plantof great commercial significance. In addition to the use of cotton fiberin the production of textiles, other uses of cotton include foodpreparation with cotton seed oil and animal feed derived from cottonseed husks.

A related goal involving the control of cell wall and characteristicswould be to affect valuable, secondary tree characteristics of wood forpaper forestry products. For instance, by altering the balance ofcellulose and lignin, the quality of wood for paper production may beimproved.

Finally, despite the importance of cotton as a crop, the breeding andgenetic engineering of cotton fiber phenotypes has taken place at arelatively slow rate because of the absence of reliable promoters foruse in selectively effecting changes in the phenotype of the fiber. Inorder to effect the desired phenotypic changes, transcription initiationregions capable of initiating transcription in fiber cells duringdevelopment are desired. Thus, an important goal of cottonbioengineering research is the acquisition of a reliable promoter whichwould permit expression of a protein selectively in cotton fiber toaffect such qualities as fiber strength, length, color and dyability.

Relevant Literature

Cotton fiber-specific promoters are discussed in PCT publications WO94/12014 and WO 95/08914, and John and Crow, Proc. Natl. Acad. Sci. USA,89:5769-5773, 1992. cDNA clones that are preferentially expressed incotton fiber have been isolated. One of the clones isolated correspondsto mRNA and protein that are highest during the late primary cell walland early secondary cell wall synthesis stages. John and Crow, supra.

In plants, control of cytoskeletal organization is poorly understood inspite of its importance for the regulation of patterns of cell division,expansion, and subsequent deposition of secondary cell wall polymers.The cotton fiber represents an excellent system for studyingcytoskeletal organization. Cotton fibers are single cells in which cellelongation and secondary wall deposition can be studied as distinctevents. These fibers develop synchronously within the boll followinganthesis, and each fiber cell elongates for about 3 weeks, depositing athin primary wall (Meinert and Delmer, (1984) Plant Physiol. 59:1088-1097; Basra and Malik, (1984) Int Rev of Cytol 89: 65-113). At thetime of transition to secondary wall cellulose synthesis, the fibercells undergo a synchronous shift in the pattern of cortical microtubuleand cell wall microfibril alignments, events which may be regulatedupstream by the organization of actin (Seagull, (1990) Protoplasma 159:44-59; and (1992) In: Proceedings of the Cotton Fiber CelluloseConference, National Cotton Council of America, Memphis RN, pp 171-192.

Agrobacterium-mediated cotton transformation is described in Umbeck,U.S. Pat. Nos. 5,004,8631 and 5,159,135 and cotton transformation byparticle bombardment is reported in WO 92/15675, published Sep. 17,1992. Transformation of Brassica has been described by Radke et al.(Theor. Appl. Genet. (1988) 75;685-694; Plant Cell Reports (1992)11:499-505.

Genes involved in lignin biosynthesis are described by Dwivedi, U. N.,Campbell, W. H., Yu, J., Datla., R. S. S., Chiang, V. L., and Podila, G.K. (1994) “Modification of lignin biosynthesis in transgenic Nicotianathrough expression of an antisense O-methyltransferase gene fromPopulus” Pl. Mol. Biol. 26: 61-71; and Tsai, C. J., Podila, G. K. andChaing, V. L. (1995) “Nucleotide sequence of Populus tremuloides genefor caffeic acid/5 hydroxyferulic acid O-methyltransferase” Pl. Physiol.107: 1459; and also U.S. Pat. No. 5,451,514 (claiming the use ofcinnamyl alcohol dehydrogenase gene in an antisense orientation suchthat the endogenous plant cinnamyl alcohol dehydrogenase gene isinhibited).

Other References Cited Throughout the Specification

1. Gibeaut, D. M., & Carpita, N. C. (1994) FASEB J. 8, 904-915.

2. Delmer, D. P., & Amor, Y. (1995) Plant cell 7, 987-1000.

3. Ross, P., Mayer, R., & Benziman, M. (1991) Microbiol. Rev. 55, 35-58.

4. Saxena, I. M., Lin, F. C., & Brown, R. M., Jr. (1990) Plant Mol.Biol. 15, 673-683.

5. Saxena, I. M., Lin, F. C., & Brown, R. M., Jr. (1992) Plant Mol.Biol. 16, 947-954.

6. Saxena, I. M., Kudlicka, K., Okuda, K., & Brown, R. M., Jr. (1994) J.Bacteriol. 176, 5735-5752.

7. Wong, H. C., Fear, A. L., Calhoon, R. D., Eidhinger, G. H., Mayer,R., Amikam, D., Benziman, M., Gelfand, D. H., Meade, J. H., Emerick, A.W., Bruner, R., Ben-Basat, B. A., & Tal, R. (1990) Proc. Natl. Acad.Sci. USA 87, 8130-8134.

8. Lin, F.-C., Brown, R. M. Jr., Drake, R. R. Jr., & Haley, B. E. (1990)J. Biol. Chem. 265, 4782-4784.

9. Mayer, R., Ross, P., Winhouse, H., Amikm, D., Volman, G., Ohana, P.,Calhoon, R. D., Wong, H. C., Emerick, A. W., & Benziman, M. (1991) Proc.Natl. Acad. Sci. USA 88, 5472-5476.

10. Matthysse, A. G., White, S., & Lightfoot, R. (1995a) J. Bacteriol.177, 1069-1075.

11. Matthysse, A. G., Thomas, D. O. L., & White, S. (1995b) J.Bacteriol. 177, 1076-1081.

12. Saxena, I. M., Brown, R. M.,Jr., Fevre, M., Geremia, R. A., &Henrissat, B. (1995) J. Bacteriol. 177, 1419-1424.

13. Meinert, M., & Delmer, D. P. (1977) Plant Physiol. 59, 1088-1097.

14. Delmer, D. P., Pear, J. R., Andrawis, A., & Stalker, D. M. (1995)Mol. Gen. Genet. 248, 43-51.

15. Delmer, D. P., Solomon, M., & Read, S. M. (1991) Plant Physiol. 95,556-563.

16. Nagai, K., & Thogersen, H. C. (1987) Methods in Enzymol. 153,461-481.

17. Laemmli, U. K. (1970) Nature 227, 680-685.

18. Kyte, J., & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132.

19. Oikonomakos, N. G., Acharya, K. R., Stuart, D. I., Melpidou, A. E.,McLaughlin, P. J., & Johnson, L. N. (1988) Eur. J. Biochem. 173,569-578.

20. Maltby, D., Carpita, N. C., Montezinos, D., Kulow, C., & Delmer, D.P. (1979) Plant Physiol. 63, 1158-1164.

21. Inoue, S. B., Takewaki, N., Takasuka, T., Mio, T., Adachi, M.,Fujii, Y., Miyamoto, C., Arisawa, M., Furuichi, Y., & Watanabe, T.(1995) Eur. J. Biochem. 231, 845-854.

22. Jacob, S. R., & Northcote, D. H. (1985) J. Cell Sci. 2 (suppl.),1-11.

23. Delmer, D. P. (1987) Annu. Rev. Plant Physiol. 38, 259-290.

24. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J.(1990) J. Mol. Biol. 215, 403-410

25. Milligan, G., Parenti, M., & Magee, A. I. (1995) TIBS 20, 183-186.

26. Amor, Y., Haigler, C. H., Johnson, S., Wainscott, M., & Delmer, D.P. (1995) Proc. Natl. Acad. Sci. USA 92, 9353-9357.

27. Amor, Y., Mayer, R., Benziman, M., & Delmer, D. P. (1991) Plant Cell3, 989-995.

SUMMARY OF THE INVENTION

Two cotton genes, CelA1 and CelA2, have been shown to be highlyexpressed in developing fibers at the onset of secondary wall cellulosesynthesis. Comparisons indicate that these genes and the rice CelA geneencode polypeptides that have three regions of reasonably high homology,both in terms of primary amino acid sequence and hydropathy, withbacterial CelA proteins. The fact that these homologous stretches are inthe same sequential order as in the bacterial CelA proteins and alsocontain four sub-regions previously predicted to be critical forsubstrate binding and catalysis (12) argues that the plant genes encodetrue homologs of bacterial CelA proteins. Furthermore, the pattern ofexpression in fiber as well as our demonstration that at least one ofthese highly-conserved regions is critical for UDP-glc binding alsosupports this conclusion.

Novel DNA promoter sequences are also supplied, and methods for theiruse are described for directing transcription of a gene of interest incotton fiber.

The developing cotton fiber is an excellent system for studies oncellulose synthesis as these single cells develop synchronously in theboll and, at the end of elongation, initiate the synthesis of a nearlypure cellulosic cell wall. During this transition period, synthesis ofother cell wall polymers ceases and the rate of cellulose synthesis isestimated to rise nearly 100-fold in vivo (13). In our continuingefforts to identify genes critical to this phase of fiber development,we have initiated a program sequencing randomly selected cDNA clonesderived from a library prepared from mRNA harvested from fibers at thestage in which secondary wall synthesis approaches its maximum rate(approximately 21 dpa).

We have characterized two cotton (Gossypium hirsutum) cDNA clones andidentified one rice (Oryza sativa) cDNA that are homologs of thebacterial CelA genes that encode the catalytic subunit of cellulosesynthase. Three regions in the deduced amino acid sequences of the plantCelA gene products are conserved with respect to the proteins encoded bybacterial CelA genes. Within these conserved regions are four highlyconserved subdomains previously suggested to be critical for catalysisand/or binding of the substrate UDP-glc. An overexpressed DNA segment ofthe cotton CelA1 gene encodes a polypeptide fragment that spans thesedomains and effectively binds UDP-glc, while a similar fragment havingone of these domains deleted does not. The plant CelA genes show littlehomology at the amino and carboxy terminal regions and also contain twointernal insertions of sequence, one conserved and one hypervariable,that are not found in the bacterial gene sequences. Cotton CelA1 andCelA2 genes are expressed at high levels during active secondary wallcellulose synthesis in the developing fiber. Genomic Southern analysesin cotton demonstrate that CelA comprises a family of approximately fourdistinct genes.

We report here the discovery of two cotton genes that showhighly-enhanced expression at the time of onset of secondary wallsynthesis in the fiber. The sequences of these two cDNA clones, termedCelA1 and CelA2, while not identical are highly homologous to each otherand to a sequenced rice EST clone discovered in the dBEST databank. Thededuced proteins also share signifigant regions of homology with thebacterial CelA proteins. Coupled with their high level and specificityof expression in fiber at the time of active cellulose synthesis, aswell as the ability of an E. coli expressed fragment of the CelA1 geneproduct to bind UDP-glc, these findings support the conclusion thatthese plant genes are true homologs of the bacterial CelA genes.

The methods of the present invention include transfecting a host plantcell of interest with a transcription or expression cassette comprisinga cotton fiber promoter and generating a plant which is grown to producefiber having the desired phenotype. Constructs and methods of thesubject invention thus find use in modulation of endogenous fiberproducts, as well as production of exogenous products and in modifyingthe phenotype of fiber and fiber products. The constructs also find useas molecular probes. In particular, constructs and methods for use ingene expression in cotton embryo tissues are considered herein. By thesemethods, novel cotton plants and cotton plant parts, such as modifiedcotton fibers, may be obtained.

The sequences and constructs of this invention may also be used toisolate related cellulose synthase genes from forest tree species, foruse in transforming and modifying wood quality. As and example, lignin,an undesirable by-product of the pulping process, by be reduced byover-expressing the cellulose synthase product and diverting productioninto cellulose.

Thus, the application provides constructs and methods of use relating tomodification of cell and cell wall phenotype in cotton fiber and woodproducts.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Northern analysis of CelA1 gene in cotton tissues and developingfiber. Approximately 10 μg total RNA from each tissue was loaded perlane. Blots were prepared and probe preparation and hybridizationconditions were performed as described previously (14). The entire CelA1cDNA insert was used as a probe in this experiment. Exposure time forthe audoradiogram was seven hours at −70°.

FIG. 2. Cotton genomic DNA analysis for both the CelA1 and CelA2 cDNAs.Approximately 10-12 μg of DNA was digested with the designatedrestriction enzymes and electrophoresed 0.9% agarose gels. Probepreparation and hybridization conditions were as described previously(14). The entire CelA1 and CelA2 cDNAs were utlized as probes. Exposuretime for the audoradiograms was three days at −70°.

FIGS. 3A, 3B, and 3C. Multiple alignment of deduced amino acid sequencesof plant and bacterial CelA proteins. (G. Hirsutum CelA-1 (SEQ ID NO:6);G. Hirsutum CelA-2 (SEQ ID NO:7); O. Sativa CelA (SEQ ID NO:8); A.Xylinum AcsAB (SEQ ID NO:9); A. Xylinum Bcsa (SEQ ID NO:10); E. coli ORFf692 (SEQ ID NO:11); and A. tumefaciens CelA (SEQ ID NO: 12)) Analyseswere performed by Clustal Analysis using the Lasergene Multalign program(DNAStar, Madison, Wis.) with gap and gap-length penalties of 10 and aPAM250 weight table. Residues are boxed and shaded when they showchemical group similarity in 4 out of 7 proteins compared. H-1, H-2, H-3regions are indicated where homology between plant and bacterialproteins is highest. The plant proteins show two insertions that are notpresent in the bacterial protein—one, P-CR, is conserved among the plantCelA genes, while a second insertion is hypervariable (HVR) betweenplant genes. The presence of the P-CR and HVR regions led to inaccuratealignments when the entire proteins were compared; the optimalalignments shown here were thus performed in five seperate blocks.Regions U-1 through U-4 are predicted to be critical for UDP-glc bindingand catalysis in bacterial CelA

FIG. 4. Kyte-Doolittle hydropathy plots of cotton CelA1 aligned withthose of two bacterial CelA proteins. Alignments and designations arebased upon those noted in FIG. 2. The hydropathy profiles shown werecalculated using a window of 7, although a window of 19 was used forpredictions of transmembrane helices that are indicated by the arrows.

FIG. 5. An E. coli expressed GST cotton CelA-1 fusion. protein binds thecontaining U1 through U4 binds UDP-glc in vitro. Panel A shows ahypothetical orientation of the cotton CelA1 protein in the plasmamembrane and indicates the cytoplasmic region containing the sub-domainsU-1 to U-4. GST-fusion constructs for CelA1 fragments spanning theregion between the potential transmembrane helices (A through H) wereprepared as described in Materials and Methods. The purified and blottedCelA1 fusion protein fragments were tested as described in Materials andMethods for their ability to bind ³²P-UDP-glc (panel B). M refers to themolecular weight markers while CS and •U1 to the full-length and deletedGST-CelA1 fusion polypeptides. The left panel shows proteins stainedwith Coomassie blue while the other three panels show representativeautoradiograms under different binding conditions as described inMaterials and Methods. Ph, BSA and Ova refer to the molecular weightstandards phosphorylase b, bovine serum albumin and ovalbuminrespectively.

FIGS. 6A, 6B, 6C and 6D. Nucleic acid sequences (SEQ ID NO: 1) to cDNAof CelA1 protein of cotton (Gossypium hirsutum).

FIGS. 7A, 7B, 7C, 7D and 7E. Nucleic acid sequences (SEQ ID NO:2) tocDNA of CelA2 protein of cotton (Gossypium hirsutum), includingapproximately the last 3′ two-thirds of the encoding region.

FIG. 8. Genomic nucleic acid sequences (SEQ ID NO:3) of CelA1 protein ofcotton (Gossypium hirsutum), including approximately 900 bases of thepromoter region 5′ to the encoding sequences.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, novel constructs and methodsare described, which may be used provide for transcription of anucleotide sequence of interest in cells of a plant host, preferentiallyin cotton fiber cells to produce cotton fiber having an altered colorphenotype.

Cotton fiber is a differentiated single epidermal cell of the outerintegument of the ovule. It has four distinct growth phases; initiation,elongation (primary cell wall synthesis), secondary cell wall synthesis,and maturation. Initiation of fiber development appears to be triggeredby hormones. The primary cell wall is laid down during the elongationphase, lasting up to 25 days postanthesis (DPA). Synthesis of thesecondary wall commences prior to the cessation of the elongation phaseand continues to approximately 40 DPA, forming a wall of almost purecellulose.

The constructs for use in such cells may include several forms,depending upon the intended use of the construct. Thus, the constructsinclude vectors, transcriptional cassettes, expression cassettes andplasmids. The transcriptional and translational initiation region (alsosometimes referred to as a “promoter,”), preferably comprises atranscriptional initiation regulatory region and a translationalinitiation regulatory region of untranslated 5′ sequences, “ribosomebinding sites,” responsible for binding mRNA to ribosomes andtranslational initiation. It is preferred that all of thetranscriptional and translational functional elements of the initiationcontrol region are derived from or obtainable from the same gene. Insome embodiments, the promoter will be modified by the addition ofsequences, such as enhancers, or deletions of nonessential and/orundesired sequences. By “obtainable” is intended a promoter having a DNAsequence sufficiently similar to that of a native promoter to providefor the desired specificity of transcription of a DNA sequence ofinterest. It includes natural and synthetic sequences as well assequences which may be a combination of synthetic and natural sequences.

Cotton fiber transcriptional initiation regions of cellulose synthaseare used in cotton fiber modification.

A transcriptional cassette for transcription of a nucleotide sequence ofinterest in cotton fiber will include in the direction of transcription,the cotton fiber transcriptional initiation region, a DNA sequence ofinterest, and a transcriptional termination region functional in theplant cell. When the cassette provides for the transcription andtranslation of a DNA sequence of interest it is considered an expressioncassette. One or more introns may be also be present.

Other sequences may also be present, including those encoding transitpeptides and secretory leader sequences as desired.

Downstream from, and under the regulatory control of, the cellulosesynthase transcriptional/translational initiation control region is anucleotide sequence of interest which provides for modification of thephenotype of fiber. The nucleotide sequence may be any open readingframe encoding a polypeptide of interest, for example, an enzyme, or asequence complementary to a genomic sequence, where the genomic sequencemay be an open reading frame, an intron, a noncoding leader sequence, orany other sequence where the complementary sequence inhibitstranscription, messenger RNA processing, for example, splicing, ortranslation. The nucleotide sequences of this invention may besynthetic, naturally derived, or combinations thereof. Depending uponthe nature of the DNA sequence of interest, it may be desirable tosynthesize the sequence with plant preferred codons. The plant preferredcodons may be determined from the codons of highest frequency in theproteins expressed in the largest amount in the particular plant speciesof interest. Phenotypic modification can be achieved by modulatingproduction either of an endogenous transcription or translation product,for example as to the amount, relative distribution, or the like, or anexogenous transcription or translation product, for example to providefor a novel function or products in a transgenic host cell or tissue. Ofparticular interest are DNA sequences encoding expression productsassociated with the development of plant fiber, including genes involvedin metabolism of cytokinins, auxins, ethylene, abscissic acid, and thelike. Methods and compositions for modulating cytokinin expression aredescribed in U.S. Pat. No. 5,177,307, which disclosure is herebyincorporated by reference. Alternatively, various genes, from sourcesincluding other eukaryotic or prokaryotic cells, including bacteria,such as those from Agrobacterium tumefaciens T-DNA auxin and cytokininbiosynthetic gene products, for example, and mammals, for exampleinterferons, may be used.

Alternatively, the present invention provides the sequences to cottoncellulose synthase, which can be expressed, or down regulated byantisense or co-suppression with its own, or other cotton or other fiberpromoters to modify fiber phenotyp.

In cotton, primary wall hemicellulose synthesis ceases as secondary wallsynthesis initiates in the fiber, and there are only two possibleβ-glucans synthesized in fibers at the time these genes arehighly-expressed; callose and cellulose (20). The following datastrongly argue against the plant CelA genes coding for callosesynthase: 1) callose synthase binds UDP-glc and is activated in aCa²⁺-dependent manner (2), while the CelA1 polypeptide fragmentcontaining the UDP-glc binding site preferentially binds UDP-glc in aMg²⁺-dependent manner, similar to bacterial cellulose synthase (9); 2)the timing of synthesis of callose in vivo in developing cotton fiber(20) does not match the expression of the cotton CelA genes (FIG. 1); 3)comparison of the CelA gene sequences with those of suspected1,3-β-glucan synthase genes from yeast (21) indicated no significanthomology.

It is still possibille that the CelA protein might encode bothactivities, as hypothesized some years ago (22-23), and the plant CelAsmight be responsible for direct polymerization of glucan from UDP-glc asproposed for A. xylinum, although they may catalyze synthesis of alipid-glc precursor as proposed for the CelA protein of A. tumefaciens.

In addition to their similarities, the plant CelA genes show severalvery interesting divergences from their bacterial ancestors, and thesemay account for the previous lack of success in using bacterial probesto detect these cDNA clones. However, a BLAST search of protein databanks (24) using the entire protein sequence of cotton CelA1 alwaysshows highest homology with the bacterial cellulose synthases. Ofparticular interest is the insertion of two unique, plant-specificregions designated P-CR and HVR. These regions are clearly not artifactsof cloning as they are observed in both cotton genes as well as the riceCelA gene. The three plant proteins show a high degree of amino acidhomology to each other throughout most of their length, diverging onlyat the N- and C-terminal ends and the very interesting HVR region. It istempting to speculate that the HVR region may confer some specificity offunction; the highly-charged and cysteine rich nature of the firstportion of HVR could make this region a potential candidate forinteraction with specific regulatory proteins, for cytoskeletalelements, or for redox regulation. In addition, we note the presence ofseveral cysteine residues near the N- and C-terminal regions of theprotein that might serve as substrates for palmytolylation and alsoserve to help anchor the protein in the membrane (25).

In summary, the finding of these plant CelA homologs potentially opensup an exciting chapter in research on cellulose synthesis in higherplants. Their finding its of particular significance since biochemicalapproaches to identification of plant cellulose synthase have provenexceedingly difficult. One obvious challenge will be to gain definitiveproof that these genes are truely functional in cellulose synthesisinvivo. Other promising goals will be to identify other components of acomplex that might interact with CelA, such as that proposed for sucrosesynthase (26), and/or a regulatory subunit that binds cyclic-di-GMP(9,27) or other glycosyltransferases (10,11).

Transcriptional cassettes may be used when the transcription of ananti-sense sequence is desired. When the expression of a polypeptide isdesired, expression cassettes providing for transcription andtranslation of the DNA sequence of interest will be used. Variouschanges are of interest; these changes may include modulation (increaseor decrease) of formation of particular saccharides, hormones, enzymes,or other biological parameters. These also include modifying thecomposition of the final fiber that is changing the ratio and/or amountsof water, solids, fiber or sugars. Other phenotypic properties ofinterest for modification include response to stress, organisms,herbicides, brushing, growth regulators, and the like. These results canbe achieved by providing for reduction of expression of one or moreendogenous products, particularly an enzyme or cofactor, either byproducing a transcription product which is complementary (anti-sense) tothe transcription product of a native gene, so as to inhibit thematuration and/or expression of the transcription product, or byproviding for expression of a gene, either endogenous or exogenous, tobe associated with the development of a plant fiber.

The termination region which is employed in the expression cassette willbe primarily one of convenience, since the termination regions appear tobe relatively interchangeable. The termination region may be native withthe transcriptional initiation region, may be native with the DNAsequence of interest, may be derived from another source. Thetermination region may be naturally occurring, or wholly or partiallysynthetic. Convenient termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. In some embodiments, it may be desired touse the 3′ termination region native to the cotton fiber transcriptioninitiation region used in a particular construct.

As described herein, in some instances additional nucleotide sequenceswill be present in the constructs to provide for targeting of aparticular gene product to specific cellular locations.

Similarly, other constitutive promoters may also be useful in certainapplications, for example the mas, Mac or DoubleMac, promoters describedin U.S. Pat. No. 5,106,739 and by Comai et al., Plant Mol. Biol. (1990)15:373-381). When plants comprising multiple gene constructs aredesired, the plants may be obtained by co-transformation with bothconstructs, or by transformation with individual constructs followed byplant breeding methods to obtain plants expressing both of the desiredgenes.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques include transfection with DNA employing A. tumefaciens or A.rhizogenes as the transfecting agent, protoplast fusion, injection,electroporation, particle acceleration, etc. For transformation withAgrobacterium, plasmids can be prepared in E. coli which contain DNAhomologous with the Ti-plasmid, particularly T-DNA. The plasmid may ormay not be capable of replication in Agrobacterium, that is, it may ormay not have a broad spectrum prokaryotic replication system such asdoes, for example, pRK290, depending in part upon whether thetranscription cassette is to be integrated into the Ti-plasmid or to beretained on an independent plasmid. The Agrobacterium host will containa plasmid having the vir genes necessary for transfer of the T-DNA tothe plant cell and may or may not have the complete T-DNA. At least theright border and frequently both the right and left borders of the T-DNAof the Ti- or Ri-plasmids will be joined as flanking regions to thetranscription construct. The use of T-DNA for transformation of plantcells has received extensive study and is amply described in EPA SerialNo. 120,516, Hoekema, In: The Binary Plant Vector SystemOffset-drukkerij Kanters B. V., Alblasserdam, 1985, Chapter V, Knauf, etal., Genetic Analysis of Host Range Expression by Agrobacterium, In:Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed.,Springer-Verlag, N.Y., 1983, p. 245, and An, et al., EMBO J. (1985)4:277-284.

For infection, particle acceleration and electroporation, a disarmedTi-plasmid lacking particularly the tumor genes found in the T-DNAregion) may be introduced into the plant cell. By means of a helperplasmid, the construct may be transferred to the A. tumefaciens and theresulting transfected organism used for transfecting a plant cell;explants maybe cultivated with transformed A. tumefaciens or A.rhizogenes to allow for transfer of the transcription cassette to theplant cells. Alternatively, to enhance integration into the plantgenome, terminal repeats of transposons may be used as borders inconjunction with a transposase. In this situation, expression of thetransposase should be inducible, so that once the transcriptionconstruct is integrated into the genome, it should be relatively stablyintegrated. Transgenic plant cells are then placed in an appropriateselective medium for selection of transgenic cells which are then grownto callus, shoots grown and plantlets generated from the shoot bygrowing in rooting medium.

To confirm the presence of the transgenes in transgenic cells andplants, a Southern blot analysis can be performed using methods known tothose skilled in the art. Expression products of the transgenes can bedetected in any of a variety of ways, depending upon the nature of theproduct, and include immune assay, enzyme assay or visual inspection,for example to detect pigment formation in the appropriate plant part orcells. Once transgenic plants have been obtained, they may be grown toproduce fiber having the desired phenotype. The fibers may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants having the desired characteristics. The termstransgenic plants and transgenic cells include plants and cells derivedfrom either transgenic plants or transgenic cells.

The various sequences provided herein may be used as molecular probesfor the isolation of other sequences which may be useful in the presentinvention, for example, to obtain related transcriptional initiationregions from the same or different plant sources. Relatedtranscriptional initiation regions obtainable from the sequencesprovided in this invention will show at least about 60% homology, andmore preferred regions will demonstrate an even greater percentage ofhomology with the probes.

Of particular importance is the ability to obtain related transcriptioninitiation control regions having the timing and tissue parametersdescribed herein. Thus, by employing the techniques described in thisapplication, and other techniques known in the art (such as Maniatis, etal., Molecular Cloning,—A Laboratory Manual (Cold Spring Harbor, N.Y.)1982), other encoding regions or transcription initiation regions ofcellulose synthase as described in this invention may be determined. Theconstructs can also be used in conjunction with plant regenerationsystems to obtain plant cells and plants; thus, the constructs may beused to modify the phenotype of fiber cells, to provide cotton fiberswhich are colored as the result of genetic engineering to heretoforunavailable hues and/or intensities.

Various varieties and lines of cotton may find use in the describedmethods. Cultivated cotton species include Gossypium hirsutum and G.babadense (extra-long stable, or Pima cotton), which evolved in the NewWorld, and the Old World crops G. herbaceum and G. arboreum.

By using encoding sequences to enzymes which control wood quality andwood product characteristics, i.e., cellulose synthase andO-methyltransferase (a key enzyme in lignin biosynthesis) the relativesynthesis of cellulose and lignin by plants may be controlled.Transformation of the plant genome with a recombinant gene constructwhich contains the gene specifying an enzyme critical to the synthesisof cellulose or lignin or a lignin precursor, in either a sense or in anantisense orientation. If an antisense orientation, the gene willtranscribed so mRNA having a sequence complementary to the equivalentmRNA transcribed from the endogenous gene is expressed, leading tosuppression of the synthesis of lignin or cellulose.

If the recombinant gene has the lignin enzyme gene in normal, or “sense”orientation, increased production of the enzyme may occur when theinsert is the full length DNA but suppression may occur if only apartial sequence is employed.

Furthermore, the expression of one maybe increased in this manner whilethe other is reduced. For instance, the production of cellulose may byincreased through the overexpression of cellulose synthase, while ligninproduction is reduced. By thus reducing the relative lignin content, thequality of wood for paper production would be improved.

EXAMPLES

The following examples are offered by way of illustration and not bylimitation.

Example 1 cDNA Libraries

An unamplified cDNA library was used to prepare the Lambda Uni-Zapvector (Stratagene, LaJolla, Calif.) using cDNA derived from polyA+ mRNAprepared from fibers of Gossypium hirsutum Acala SJ-2 harvested at 21DPA, the time at which secondary wall cellulose synthesis is approachinga maximal rate (13). Approximately 250 plaques were randomly selectedfrom the cDNA library, phages purified and plasmids excised from thephage vector and transformed.

The resulting clones/inserts were size screened on 0.8% agarose gels(DNA inserts below 600 bp were excluded).

Example 2 Isolation and Sequencing of cDNA Clones

Plasmid DNA inserts were randomly sequenced using an Applied Biosystems(Foster City, Calif.) Model 373A DNA sequencer. A search of the GenBankEST databank revealed that there were at least 23 rice and 8 ArabidopsisEST clones that contain sequences similar to the cotton CelA1 DNAsequence. EST clone S14965 was obtained from Y. Nagamura (Rice GenomeResearch Program, Tsukuba). A series of deletion mutants were generatedand used for DNA sequencing analysis at the Weizmann Institute ofScience (Rehovot).

Example 3 Northern and Southern Analyses.

Cotton plants (G. hirsutum cv. Coker 130) were grown in the greenhouseand tissues harvested at the appropriate times indicated and frozen inliquid N₂. Total cotton RNA and cotton genomic DNA was prepared andsubjected to Northern and Southern analyses as described previously(14).

Example 4 UDP-Glc Binding Studies

To construct a GST-CelA1 protein fusion, a 1.6 kb DNA CelA1 DNA fragmentcontaining a putative cytoplasmic domain between the second and thirdtransmembrane helices was PCR amplified with the primersATTGAATTCCTGGGTGTTGGATCAGTT (SEQ ID NO:4) and ATTCTCGAGTGGAAGGGATTGAAA(SEQ ID NO: 5) in a reaction containing 1 ng plasmid DNA (clone 213) astemplate. The amplified fragment was unidirectionally cloned into theEcoRI and XhoI sites of the GST expression vector pGEX4T-3 (Pharmacia),generating a fusion protein GST-CS containing the amino acids Ser215 toLeu759 of the cotton CelA1 protein. Two CelA1 gene internal PstI siteswithin the plasmid pGST-CS were used to generate the deletion mutantpGST-CSΔU1, which lacks 196 amino acids (and the U1 binding region) fromVal252 to Ala447.

For the UDGP binding assays, α-³²P-labeled UDP-glc was prepared asdescribed (15). The two fusion proteins GST-CS and GST-CS·U1 wereexpressed in E. coli and purified from inclusion bodies (16). Proteinswere suspended in sample buffer, heated to 100° C. for 5 min andapproximately 50 ng of the two fusion protein products and molecularweight standards (Bio-Rad) subjected to SDS-PAGE using 4.5% and 7.5%acrylamide in the stacking and separating gels, respectively (17). Afterelectrophoresis, protein transfer to nitrocellulose filters was carriedout in transfer buffer (25 mM Tris, 192 mM glycine and 20% (v/v)methanol). The filter was briefly rinsed in deionized H₂O and incubatedin PBS buffer for 15 min, then stained with Ponceau-S in PBS buffer.After washing in deionized H₂O, protein was further renatured on thefilter by incubation in PBS buffer for 30 min and used directly forbinding assays. All binding buffers contained 50 mM HEPES/KOH (pH 7.3),50 mM NaCl and 1 mMDTT. In addition, binding buffers contained either 5mM MgCl₂ and 5 mM EGTA (Buffer Mg/EGTA), 5 mM EDTA (Buffer EDTA) or 1 mMCaCl₂ and 20 mM cellobiose (Buffer Ca/CB). Binding reaction was carriedout in 7 ml containing ³²P-labeled UDP-glc (1×10⁷ cpm) at roomtemperature for 3 hours with constant shaking. Filters were washedseparately three times in 20 ml washing buffer consisting of 50 mMHEPES/KOH (pH 7.3) and 50 mM NaCl for 5 min each, briefly dried andanalyzed on a Bio-imaging analyzer BAS1000 (Fugi).

Example 5 Identification, Differential Expression and Genomic Analysisof Cotton CelA Genes

During the course of screening and sequencing random cDNA clones from acotton fiber specific cDNA library prepared from RNA collectedapproximately 21 dpa, it was discovered that two cDNA clones thatinitially exhibited small blocks of amino acid homology to the proteinsencoded by the bacterial CelA genes. Clone 213 appeared to befull-length cDNA while another distinct clone, 207, appeared to be apartial clone relative to the length of 213. These two clones werepartially homologous at the nucleotide and amino acid levels anddesignated CelA1 and CelA2 respectively.

These clones were then utilized as probes for Northern blot analysis todetermine their differential expression in cotton tissues and developingcotton fiber. FIG. 1 indicates the expression pattern for the CelA1gene. The CelA1 gene encodes a mRNA of approximately 3.2 kb in lengthand is expressed at extremely high levels in developing fiber, beginningat approximately 17 dpa, the time at which secondary wall cellulosesynthesis is initiated(13). The gene is also expressed at low levels inall other cotton tissues, most notably in root, flower and developingseeds. Since regions of these genes are somewhat homologous at thenucleotide level, gene specific probes were designed (using thehypervariable regions described in FIG. 3) to distinguish the specificexpression patterns of CelA1 and CelA2. These gene specific probesgenerated expression patterns (data not shown) for the two genesidentical to that shown in FIG. 1, except that a very low mRNA level wasalso detected in the primary wall phase of fiber development (5-14 dpa)for the CelA2 gene when the blots were overexposed. The CelA2 genespecific probe also encoded a 3.2 kb mRNA, analogous in size to the mRNAspecified by the gene for CelA1. Messenger RNAs for both genes exhibit acharacteristic degradation pattern similar to other mRNAs specificallyexpressed late in fiber development (J. Pear, unpublished observations)and this degradation is not a result of the integrity of the mRNApreparations (14). We estimate that both cotton CelA genes are expressedin developing fiber approximately 500 times their level of expression inother cotton tissues and that they constitute approximately 1-2% of the24 dpa fiber mRNA.

In order to estimate the number of CelA genes in the cotton genome,Southern analysis was performed utilizing both CelA cDNAs independentlyas probes (FIG. 2). Although the two cotton genes are fairlynon-homologous at the nucleotide level over their entire length, thereare regions of homology (the H1, H2 and H3 regions described below) andit was thought these regions could be useful in identifying other cottonCelA genes. FIG. 2 indicates that the CelA1 cDNA probe will hybridize,albeit weakly, to the CelA2 genomic equivalent and vise versa. TheHindIII pattern for both genes and cDNA probes is particularlydiscriminating. There are also a number of other weakly hybridzing bandsin these digests and from these data we estimate that the cotton CelAgenes constitute a small family of approximately four genes. Homology ofPlant and Bacterial CelA Gene Products.

In addition to the two similar cotton CelA genes, a homologous cDNAclone was discovered in the dBest databank of rice and Arabidopsis ESTs.Accession No. D48636, the rice clone having the longest insert wasobtained and sequenced, and the homology comparisons with bacterialproteins reported here also include results with the rice CelA. FIGS.3A, 3B and 3C shows the results of a multiple alignment of the deducedamino acid sequences from the three plant CelA genes and four bacterialCelA genes from A. xylinum (AcsAB and BcsA), E. coli, and A.tumefaciens. FIG. 4 shows hydropathy plots (18) of cotton CelA1similarly aligned with two bacterial CelA proteins and serves as a moregeneral summary of the overall homologies.

*The following accession numbers were identified as showing homologywith cotton CelA-1. For rice: D48636, D41261, D4069111, D46824, D47622,D47175, D41766, D41986, D24655, D23732, D24375, D47732, D47821, D47850,D47494, D24964, D24862, D24860, D24711, D23841, D48053, D48612, D40673;for Arabidopsis: T45303, T45414, H76149, H36985, Z30729, H36425, T45311,A35212.

Of the plant genes, only the cotton CelA1 appears to be a full-lengthclone of 3.2 kb exhibiting an open reading frame that could potentiallycode for a polypeptide of 109,586 kD, a pI of 6.4, and four potentialsites of N-glycosylation. Comparison of the N-terminal region of cottonCelA1 with bacterial genes indicates that the plant protein has anextended N-terminal similar in length and hydropathy profile, but withonly poor amino acid sequence homology to the A. tumefaciens CelAprotein. In general, sequence homology of plant and bacterial genes inboth the N-terminal and C-terminal regions is poor. However, althoughoverall similarity comparing plant to bacterial proteins is less than25%, three homologous regions were identified, called H-1, H-2, and H-3,where the sequence similarity rises to 50-60% at the amino acid level.Interspersed between these regions of homology are two plant-specificregions not found at all in the bacterial proteins. Sequences in thefirst of these insertions are highly conserved in the plant genes(P-CR), while the second interspersed region seems to be a hypervariableregions (HVR) for there is considerable sequence divergence among theplant proteins analyzed.

None of the plant or bacterial CelA proteins contains obvious signalsequences even though they are presumably transmembrane proteins (4).However, the overall profiles suggest two potential transmembranehelices in the N-terminal and six in the C-terminal region of the cottonCelA1 that could anchor the protein in the membrane (see arrows FIGS.3A, 3B and 3C and also panel A of FIG. 5). The amino acid sequencepositions for these predicted transmembrane helices are: A (169-187), B(200-218), C (759-777), D (783-801), E (819-837), F (870-888), G(903-921), H (933-951). The central portions of the proteins are morehydrophilic and are predicted to reside in the cytoplasm and contain thesite(s) of catalysis. More detailed inspection of these hydrophilicstretches reveals four particularly conserved sub-regions (marked U-1through U-4 on FIGS. 3A, 3B and 3C and FIG. 4) that contain theconserved asp (D) residues (in U-1-3) and the motif QXXRW (in U-4) thathave been proposed (12) to be involved in substrate binding and/orcatalysis.

Binding of UDP-glucose. Further evidence that the proteins encoded bythese plant genes are CelA homologs comes from our demonstration that aDNA segment encoding the central region of the cotton CelA1 protein,over-expressed in E. coli, binds UDP-glc. We subcloned a 1.6 kb fragmentof the cotton CelA1 clone to create a hybrid gene that encodes GST fusedto the CelA1 sequence encoding amino acid residues 215-759 of the CelA1protein (FIG. 5a). This region spans U-1 through U-4 that are suspectedto be critical for UDP-glc binding. As a control, another GST fusion wascreated using a 1.0 kb PstI fragment that had the U-1 region deleted andmight not be predicted to bind UDP-glc. The fusion proteins wereoverexpressed in E. coli, purifed, and shown to have the predicted sizesof approximately 87 and 64 kD, respectively (FIG. 5b). The purifiedproteins were then subjected to SDS-PAGE, and blotted to nitrocellulose.Blotted proteins were renatured, and incubated with ³²P-UDP-glc in orderto test for binding (FIG. 5b). As predicted, the 87 kD GST-CelA1 fusiondoes indeed bind UDP-glc in a Mg²⁺ dependent manner, while the shorterfusion with the U-1 domain deleted did not show any binding (Althoughnot observed in the experiment shown, in some experiments very weaklabeling in the presence of Ca²⁺ could be observed). As furthercontrols, note that the molecular weight standards BSA and ovalbumin,proteins lacking UDP-glc binding sites, show no interaction withUDP-glc, while phosphorylase b, an enzyme inhibited by UDP-glc (19),binds this substrate.

FIGS. 6A, 6B, 6C and 6D provide the encoding sequence to the cDNA tocelA1 (start ATG at˜base 179), while FIGS. 7A, 7B, 7C, 7D and 7E providethe encoding sequence to the approximately two-thirds 3′ of the cDNA tocelA2.

Example 6 Genomic DNA

cDNA for the cellulose synthase clones was used to probe for genomicclones. For both, full length genomic DNA was obtained from a librarymade using the lambda dash 2 vector from Stratagene™, which was used toconstruct a genomic DNA library from cotton variety Coker 130 (Gossypiumhirsutum cv. coker 130), using DNA obtained from germinating seedlings.

The cotton genomic library was probed with a cellulose synthase probeand genomic phage candidates were identified and purified. FIG. 8provides an approximately 1 kb sequence of the cellulose synthasepromoter region which is immediately 5′ to the celA1 encoding region.The start of the cellulose synthase enzyme encoding region is at the ATGat base number 954.

Example 7 Cotton Transformation

Explant Preparation

Promoter constructs comprising the cellulose synthase promoter sequencesof celA1 can be cotton prepared. Coker 315 seeds are surface disinfectedby placing in 50% Clorox (2.5% sodium hypochlorite solution) for 20minutes and rinsing 3 times in sterile distilled water. Followingsurface sterilization, seeds are germinated in 25×150 sterile tubescontaining 25 mls ½×MS salts: ½×B5 vitamins: 1.5% glucose: 0.3% gelrite.Seedlings are germinated in the dark at 28° C. for 7 days. On theseventh day seedlings are placed in the light at 28±2° C.

Cocultivation and Plant Regeneration

Single colonies of A. tumefaciens strain 2760 containing binary plasmidspCGN2917 and pCGN2926 are transferred to 5 ml of MG/L broth and grownovernight at 30° C. Bacteria cultures are diluted to 1×10⁸ cells/ml withMG/L just prior to cocultivation. Hypocotyls are excised from eight dayold seedlings, cut into 0.5-0.7 cm sections and placed onto tobaccofeeder plates (Horsch et al. 1985). Feeder plates are prepared one daybefore use by plating 1.0 ml tobacco suspension culture onto a petriplate containing Callus Initiation Medium CIM without antibiotics (MSsalts: B5 vitamins: 3% glucose: 0.1 mg/L 2,4-D: 0.1 mg/L kinetin: 0.3%gelrite, pH adjusted to 5.8 prior to autoclaving). A sterile filterpaper disc (Whatman #1) was placed on top of the feeder cells prior touse. After all sections are prepared, each section was dipped into an A.tumefaciens culture, blotted on sterile paper towels and returned to thetobacco feeder plates.

Following two days of cocultivation on the feeder plates, hypocotylsections are placed on fresh Callus Initiation Medium containing 75 mg/Lkanamycin and 500 mg/L carbenicillin. Tissue is incubated at 28±2° C.,30 uE 16:8 light:dark period for 4 weeks. At four weeks the entireexplant is transferred to fresh callus initiation medium containingantibiotics. After two weeks on the second pass, the callus is removedfrom the explants and split between Callus Initiation Medium andRegeneration Medium (MS salts: 40 mM KNO₃: 10 mM NH₄Cl:B5 vitamins:3%glucose:0.3% gelrite:400 mg/L carb:75 mg/L kanamycin).

Embryogenic callus is identified 2-6 months following initiation and wassubcultured onto fresh regeneration medium. Embryos are selected forgermination, placed in static liquid Embryo Pulsing Medium (Stewart andHsu medium: 0.01 mg/l NAA: 0.01 mg/L kinetin: 0.2 mg/L GA3) andincubated overnight at 30° C. The embryos are blotted on paper towelsand placed into Magenta boxes containing 40 mls of Stewart and Hsumedium solidified with Gelrite. Germinating embryos are maintained at28±2° C. 50 uE m⁻²s⁻¹ 16:8 photoperiod. Rooted plantlets are transferredto soil and established in the greenhouse.

Cotton growth conditions in growth chambers are as follows: 16 hourphotoperiod, temperature of approximately 80-85°, light intensity ofapproximately 500 μEinsteins. Cotton growth conditions in greenhousesare as follows: 14-16 hour photoperiod with light intensity of at least400 μEinsteins, day temperature 90-95° F., night temperature 70-75° F.,relative humidity to approximately 80%.

Plant Analysis

Flowers from greenhouse grown Tl plants are tagged at anthesis in thegreenhouse. Squares (cotton flower buds), flowers, bolls etc. areharvested from these plants at various stages of development and assayedfor observable phenotype or tested for enzyme activity.

Example 7 Transformation of Tree Species

Numerous methods are known to the art for transforming forest treespecies, for example U.S. Pat. No. 5,654,190 discloses a process forproducing transgenic plant belonging to the genus Populus, the sectionLeuce.

The above results demonstrate how the cellulose synthase cDNA may beused to alter the phenotype of a transgenic plant cell, and how thepromoter may be used to modify transgenic cotton fiber cells.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application are specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail, byway of illustration and example for purposes of clarity andunderstanding, it will be readily apparent to those of ordinary skill inthe art that certain changes and modifications may be made thereto,without departing from the spirit or scope of the appended claims.

12 1 3328 DNA Artificial Sequence Synthetic Oligonucleotide 1 cgaaattaaccctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc 60 tagaactagtggatcccccg ggctgcagga attcggcacg agggttagca tattgtttgt 120 agcattgggtttttttctca aggaagaaga aggagaaaga taagtacttt ttttgagaat 180 gatggaatctggggttcctg tttgccacac ttgtggtgaa catgttgggt tgaatgttaa 240 tggtgaaccttttgtggctt gccatgaatg taatttccct atttgtaaga gttgttttga 300 gtatgatcttaaggaaggac gaaaagcttg cttgcgttgt ggtagtccat atgatgaaaa 360 cctgttggacgatgtcgaga aggccaccgg cgatcaatcg acaatggctg cacatttgaa 420 caagtctcaggatgttggaa ttcatgcaag acatatcagc agtgtgtcta cattggatag 480 tgaaatggctgaagacaatg ggaattcgat ttggaagaac agggtggaaa gttggaaaga 540 aaagaagaacaagaagaaga agcctgcaac aactaaggtt gaaagagagg ctgaaatccc 600 acctgagcaacaaatggaag ataaaccggc accggatgct tcccagcccc tctcgactat 660 aattccaatcccgaaaagca gacttgcacc ataccgaacc gtgatcatta tgcgattgat 720 cattcttggtcttttcttcc attatcgagt aacaaacccc gttgacagtg cttttggact 780 gtggctcacttcagtcatat gtgaaatctg gtttgcattt tcctgggtgt tggatcagtt 840 ccctaagtggtatcctgtta acagggaaac atacattgac agactatctg caagatatga 900 aagagaaggtgaacctgatg aacttgctgc agttgacttc ttcgtgagta cagtggatcc 960 attgaaagagcctccattga ttactgccaa tactgtgctt tccatccttg ccttggacta 1020 cccggtggataaggtctctt gttatatatc tgatgatggt gcggccatgc tgacatttga 1080 atctctagtagaaacagccg actttgcaag aaagtgggtt ccattctgca aaaaattttc 1140 cattgaaccccgggcacctg agttttactt ctcacagaag attgattact tgaaagataa 1200 agtgcagccctcttttgtaa aagaacgtag agctatgaaa agagattatg aagagtacaa 1260 aattcgaatcaatgctttag ttgcaaaggc tcagaaaaca cctgatgaag gatggacaat 1320 gcaagatggaacttcttggc caggaaataa cccgcgtgat caccctggca tgattcaggt 1380 tttccttggatatagtggtg ctcgtgacat cgaaggaaat gaacttcctc gactggttta 1440 cgtctctagagagaagagac ctggctacca acaccacaaa aaggctggtg ctgaaaatgc 1500 tttggttagggtgtctgcag ttcttacaaa tgctcccttc atcctcaatc ttgattgtga 1560 ccactatgttaacaatagca aggcagttag ggaggcaatg tgcttcttga tggacccaca 1620 agttggtcgagatgtatgct atgtgcagtt tcctcaaaga tttgatggca tagataggag 1680 tgatcgatatgccaatagga acacagtttt ctttgatgtt aacatgaaag gtcttgatgg 1740 aatccaagggccagtttatg tgggaacagg ttgtgttttc aataggcaag cactttatgg 1800 ctatggtccaccttcaatgc caagttttcc caagtcatcc tcctcatctt gctcgtgttg 1860 ctgcccgggcaagaaggaac ctaaagatcc atcagagctt tatagggatg caaaacggga 1920 agaacttgatgctgccatct ttaaccttag ggaaattgac aattatgatg agtatgaaag 1980 atcaatgttgatctctcaaa caagctttga gaaaactttt ggcttatctt cagtcttcat 2040 tgaatctacactaatggaga atggaggagt ggctgaatct gccaaccctt ccacactaat 2100 caaggaagcaattcatgtca tcagctgtgg ctatgaagag aagactgcat gggggaaaga 2160 gattggatggatatatggtt cagtcactga ggatatctta accggcttca aaatgcactg 2220 ccgaggatggagatcgattt actgcatgcc cttaaggcca gcattcaaag gatctgcacc 2280 catcaatctgtctgatcggt tgcaccaggt tcttcgatgg gctcttggat ctgttgaaat 2340 tttcctaagcaggcattgcc ctctatggta tggctttgga ggtggtcgtc ttaaatggct 2400 tcaaagactagcatatataa acaccattgt ctatcctttc acatcccttc cactcattgc 2460 ctattgttcactaccagcaa tctgtcttct cacaggaaaa tttatcatac caacgctctc 2520 aaacctggcaagtgttctct ttcttggcct tttcctttcc attatcgtga ctgctgttct 2580 cgagctccgatggagtggtg tcagcattga ggacttatgg cgtaacgagc agttttgggt 2640 catcggtggcgtttcagccc atctctttgc cgtcttccaa ggtttcctta agatgcttgc 2700 gggcattgacaccaacttta ctgtcactgc caaagcagct gatgatgcag attttggtga 2760 gctctacattgtgaaatgga ctacacttct aatccctcca acaacactcc tcatcgtcaa 2820 catggttggtgtcgttgccg gattctccga tgccctcaac aaagggtacg aagcttgggg 2880 accactctttggcaaagtgt tcttttcctt ctgggtcatc ctccatcttt atccattcct 2940 caaaggtcttatgggacgcc aaaacaggac accaaccatt gttgtccttt ggtcagtgtt 3000 gttggcttctgtcttctctc ttgtttgggt tcggatcaac ccgtttgtca gcaccgccga 3060 tagcaccaccgtgtcacaga gctgcatttc cattgattgt tgatgatatt atgtgtttct 3120 tagaattgaaatcattgcaa gtaagtggac tgaaacatgt ctattgacta agttttgaac 3180 agtttgtacccattttattc ttagcagtgt gtaattttcc taaacaatgc tatgaactat 3240 acatatttcattgatattta cattaaatga aactacatca gtctgcagaa aaaaaaaaaa 3300 aaaaaaaaactcgagggggg gcccggta 3328 2 4612 DNA Artificial Sequence SyntheticOligonucleotide 2 aactagtgga tcccccgggc tgcaggaatt cggcacgagc gaggagatgggttccgtttt 60 gtaagaagca ttgatcacct agggggcccg acgtccttaa gccgtgctcgctcctctacc 120 caaggcaaaa cattcttcgt taatgttgag cccagggcgc cggagttttatttcaatgag 180 aagattgatt atttgaagga caaggtccat attacaactc gggtcccgcggcctcaaaat 240 aaagttactc ttctaactaa taaacttcct gttccaggta cctagctttgttaaagaacg 300 gagagccatg aaaagggaat atgaagaatt taaagtaagg atcaatgcatggatcgaaac 360 aatttcttgc ctctcggtac ttttccctta tacttcttaa atttcattcctagttacgta 420 tagtagcaaa agctcagaag aaaccagaag aaggatgggt gatgcaagatggcaccccat 480 ggcccggaaa atcatcgttt tcgagtcttc tttggtcttc ttcctacccactacgttcta 540 ccgtggggta ccgggccttt taacactcgt gatcatcctg gaatgattcaggtctatcta 600 ggaagtgccg gtgcactcga tgtggatggc attgtgagca ctagtaggaccttactaagt 660 ccagatagat ccttcacggc cacgtgagct acacctaccg aaagagctgcctcgacttgt 720 ctatgtttct cgtgagaaac gacctggtta tcagcaccat aagaaagccgtttctcgacg 780 gagctgaaca gatacaaaga gcactctttg ctggaccaat agtcgtggtattctttcggc 840 gtgctgagaa tgctctggtt cgagtttctg cagtgcttac taatgcacccttcatattga 900 atctggattg cacgactctt acgagaccaa gctcaaagac gtcacgaatgattacgtggg 960 aagtataact tagacctaac tgatcattac atcaacaata gcaaggccatgagggaagcg 1020 atgtgctttt taatggatcc tcagtttgga actagtaatg tagttgttatcgttccggta 1080 ctcccttcgc tacacgaaaa attacctagg agtcaaacct aagaagctttgttatgttca 1140 atttccacag agatttgatg gtattgatcg tcatgatcga tatgctaatcttcttcgaaa 1200 caatacaagt taaaggtgtc tctaaactac cataactagc agtactagctatacgattag 1260 gaaatgttgt cttctttgat atcaacatgt tgggattaga tggacttcaaggccctgtat 1320 atgtaggcac ctttacaaca gaagaaacta tagttgtaca accctaatctacctgaagtt 1380 ccgggacata tacatccgtg agggtgtgtt ttcaacaggc aggcattgtatggctacgat 1440 ccaccagtct ctgagaaacg accaaagatg tcccacacaa aagttgtccgtccgtaacat 1500 accgatgcta ggtggtcaga gactctttgc tggtttctac acatgtgattgctggccttc 1560 ttggtgttgc tgttgttgcg gaggttctag gaagaaatca aagaagaaagtgtacactaa 1620 cgaccggaag aaccacaacg acaacaacgc ctccaagatc cttctttagtttcttctttc 1680 gtgaaaagaa gggcttactc ggaggtcttt tatacggaaa aaagaagaagatgatgggca 1740 aaaactatgt cacttttctt cccgaatgag cctccagaaa atatgccttttttcttcttc 1800 tactacccgt ttttgataca gaaaaaaggg tctgcaccag tctttgatctcgaagaaatc 1860 gaagaagggc ttgaaggata cgaagaattg cttttttccc agacgtggtcagaaactaga 1920 gcttctttag cttcttcccg aacttcctat gcttcttaac gagaaatcgacattaatgtc 1980 gcagaagaat ttcgagaaac gattcggaca atcaccggtt ttcattgcctctctttagct 2040 gtaattacag cgtcttctta aagctctttg ctaagcctgt tagtggccaaaagtaacgga 2100 caactttgat ggaaaatggt ggccttcctg aaggaactaa ttccacatcactgattaaag 2160 aggccattca gttgaaacta ccttttacca ccggaaggac ttccttgattaaggtgtagt 2220 gactaatttc tccggtaagt cgtaattagc tgtggttatg aagaaaaaactgagtggggc 2280 aaagagatcg gatggattta tgggtcggtg gcattaatcg acaccaatacttcttttttg 2340 actcaccccg tttctctagc ctacctaaat acccagccac acggaagatatattaacagg 2400 tttcaagatg cattgtagag ggtggaaatc ggtttattgt gtaccgaaaatgccttctat 2460 ataattgtcc aaagttctac gtaacatctc ccacctttag ccaaataacacatggctttt 2520 gaccggcatt caaagggtcc gctccaatca atctctcgga tcggttgcaccaagttttga 2580 gatgggcact ctggccgtaa gtttcccagg cgaggttagt tagagagcctagccaacgtg 2640 gttcaaaact ctacccgtga tggttctgta gaaattttcc ttagtcgtcactgtccactt 2700 tggtatggtt atggtggaaa actgaaatgg accaagacat ctttaaaaggaatcagcagt 2760 gacaggtgaa accataccaa taccaccttt tgactttacc ctcgagaggcttgcttatat 2820 caacaccatt gtttaccctt tcacctcgat ccctttactc gcctattgtagagctctccg 2880 aacgaatata gttgtggtaa caaatgggaa agtggagcta gggaaatgagcggataacat 2940 ctattccagc tgtttgtctt ctcaccggca aattcatcat tccaactctaagcaacctta 3000 caagtgtgtg gataaggtcg acaaacagaa gagtggccgt ttaagtagtaaggttgagat 3060 tcgttggaat gttcacacac gttcttggca cttttcctct ccatcattgcaactggagtg 3120 cttgaacttc gatggagcgg ggttagcatc caagaaccgt gaaaaggagaggtagtaacg 3180 ttgacctcac gaacttgaag ctacctcgcc ccaatcgtag caagactggtggcgcaatga 3240 acaattctgg gtgatcggag gtgtctccgc ccatcttttt gctgtcttccgttctgacca 3300 ccgcgttact tgttaagacc cactagcctc cacagaggcg ggtagaaaaacgacagaagg 3360 agggcctcct caaagtccta gctggagtag acaccaactt caccgtaacagcaaaagcag 3420 cagacgatac tcccggagga gtttcaggat cgacctcatc tgtggttgaagtggcattgt 3480 cgttttcgtc gtctgctatg agaattcggt gaactttatc tcttcaaatggacaactctc 3540 ttaatccctc ccacaactct gataatactg tcttaagcca cttgaaatagagaagtttac 3600 ctgttgagag aattagggag ggtgttgaga ctattatgac aacatggtcggagtcgtggc 3660 cggagtttca gacgcaatca acaacggcta tggttcatgg ggtccattgtttgtaccagc 3720 ctcagcaccg gcctcaaagt ctgcgttagt tgttgccgat accaagtaccccaggtaaca 3780 tcggcaaact gttcttcgca ttctgggtca ttcttcatct ttacccattcctcaaaggtt 3840 tgatggggag agccgtttga caagaagcgt aagacccagt aagaagtagaaatgggtaag 3900 gagtttccaa actacccctc acaaaacagg acgcccacca ttgttgtgctttggtccata 3960 cttttggcat cgattttctc actggtttgg tgttttgtcc tgcgggtggtaacaacacga 4020 aaccaggtat gaaaaccgta gctaaaagag tgaccaaacc gtacggatcgatcccttctt 4080 gcccaaacaa acaggtccag ttcttaaaca atgtggcgtg gagtgctaaacatgcctagc 4140 tagggaagaa cgggtttgtt tgtccaggtc aagaatttgt tacaccgcacctcacgattt 4200 tggtgtttta caaacctttc ttattatttt attttccctt tttgccactactgttgattt 4260 gctgtgattc accacaaaat gtttggaaag aataataaaa taaaagggaaaaacggtgat 4320 gacaactaaa cgacactaag taaaagggat ttatcttgtt tgtaaaaagtctcctatgat 4380 tttgttggtt caatttaatt tctatatggt attttcccta aatagaacaaacatttttca 4440 gaggatacta aaacaaccaa gttaaattaa agatatacca aaaaaaatatttctttaaat 4500 taactataaa aaaaaaaaaa aaaaactcga gggggggccc ggtacctttttttataaaga 4560 aatttaattg atattttttt tttttttttt tgagctcccc cccgggccatgg 4612 3 1063 DNA Artificial Sequence Synthetic Oligonucleotide 3gggtgattga ctaaaatttt taaaaatttt gaaggtttta atgagaattt ttaaacaatt 60ttgtatgtta aactaaaact ttcaaaaaaa attttgaaag gtttaatgag aattttaaaa 120attttgagcg ggctaattaa aatttttaaa aaatgtataa taaaaaaatt caaaaactct 180ttgaggccat aaaggtcatc gggcccttaa atacatcagc ttgttgtttc ctcatattac 240tcatgttatt tcagttaaca gatataatgg ctatcatttg atttaggagt gaaatctaaa 300aattcgaaaa gtataaaaac taaaaaggat taaattgaag aacattaatt aaatcaacaa 360tttactattc caataacaga attttgagtt aacaaattta actgctacaa tttggttcga 420gaccaaaatt acaaaacccg aaaagtattg ggactaaaat tgatcaaatt agagtacatg 480ggttaaattc acaacttact tatggtacaa ggattaatag cataatttct ccttaggcaa 540atgccagtta gttaaagatg taccttgccc aaccgaaagc ttccttaaac ttcccgcaat 600tttttaaatt tctttttccc ttagaaaaaa gaacaaaaat gtaagctttg cttgtcagag 660atttctctgc aaatacattg acaccaacaa cctaccctcc attacactac caaccggcct 720tccccttcaa cttttcttca ccattacaac atgcctatct ccacccttag cccaacatgc 780acttatatct tgtgtttggt tgtttttctt tttcatataa aaacacacac caagacacaa 840aggtattgag aggtaagtag agggaaagac cctttggtta gcatattgtt tgtagcattg 900ggttttttct caaggaagaa gaaggagaaa gataagtact ttttttgaga atgatggaat 960ctggggttcc tgtttgccac acttgtggtg aacatgttgg gttgaatgta agccgaattc 1020cagcacactg gcggccgtta ctagtggatc cgcgctcggt acc 1063 4 27 DNA ArtificialSequence Synthetic Oligonucleotide 4 attgaattcc tgggtgttgg atcagtt 27 524 DNA Artificial Sequence Synthetic Oligonucleotide 5 attctcgagtggaagggatt gaaa 24 6 974 PRT Gossypim hirsutum 6 Met Met Glu Ser Gly ValPro Val Cys His Thr Cys Gly Glu His Val 1 5 10 15 Gly Leu Asn Val AsnGly Glu Pro Phe Val Ala Cys His Glu Cys Asn 20 25 30 Phe Pro Ile Cys LysSer Cys Phe Glu Tyr Asp Leu Lys Glu Gly Arg 35 40 45 Lys Ala Cys Leu ArgCys Gly Ser Pro Tyr Asp Glu Asn Leu Leu Asp 50 55 60 Asp Val Glu Lys AlaThr Gly Asp Gln Ser Thr Met Ala Ala His Leu 65 70 75 80 Asn Lys Ser GlnAsp Val Gly Ile His Ala Arg His Ile Ser Ser Val 85 90 95 Ser Thr Leu AspSer Glu Met Ala Glu Asp Asn Gly Asn Ser Ile Trp 100 105 110 Lys Asn ArgVal Glu Ser Trp Lys Glu Lys Lys Asn Lys Lys Lys Lys 115 120 125 Pro AlaThr Thr Lys Val Glu Arg Glu Ala Glu Ile Pro Pro Glu Gln 130 135 140 GlnMet Glu Asp Lys Pro Ala Pro Asp Ala Ser Gln Pro Leu Ser Thr 145 150 155160 Ile Ile Pro Ile Pro Lys Ser Arg Leu Ala Pro Tyr Arg Thr Val Ile 165170 175 Ile Met Arg Leu Ile Ile Leu Gly Leu Phe Phe His Tyr Arg Val Thr180 185 190 Asn Pro Val Asp Ser Ala Phe Gly Leu Trp Leu Thr Ser Val IleCys 195 200 205 Glu Ile Trp Phe Ala Phe Ser Trp Val Leu Asp Gln Phe ProLys Trp 210 215 220 Tyr Pro Val Asn Arg Glu Thr Tyr Ile Asp Arg Leu SerAla Arg Tyr 225 230 235 240 Glu Arg Glu Gly Glu Pro Asp Glu Leu Ala AlaVal Asp Phe Phe Val 245 250 255 Ser Thr Val Asp Pro Leu Lys Glu Pro ProLeu Ile Thr Ala Asn Thr 260 265 270 Val Leu Ser Ile Leu Ala Leu Asp TyrPro Val Asp Lys Val Ser Cys 275 280 285 Tyr Ile Ser Asp Asp Gly Ala AlaMet Leu Thr Phe Glu Ser Leu Val 290 295 300 Glu Thr Ala Asp Phe Ala ArgLys Trp Val Pro Phe Cys Lys Lys Phe 305 310 315 320 Ser Ile Glu Pro ArgAla Pro Glu Phe Tyr Phe Ser Gln Lys Ile Asp 325 330 335 Tyr Leu Lys AspLys Val Gln Pro Ser Phe Val Lys Glu Arg Arg Ala 340 345 350 Met Lys ArgAsp Tyr Glu Glu Tyr Lys Ile Arg Ile Asn Ala Leu Val 355 360 365 Ala LysAla Gln Lys Thr Pro Asp Glu Gly Trp Thr Met Gln Asp Gly 370 375 380 ThrSer Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met Ile Gln 385 390 395400 Val Phe Leu Gly Tyr Ser Gly Ala Arg Asp Ile Glu Gly Asn Glu Leu 405410 415 Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Tyr Gln His420 425 430 His Lys Lys Ala Gly Ala Glu Asn Ala Leu Val Arg Val Ser AlaVal 435 440 445 Leu Thr Asn Ala Pro Phe Ile Leu Asn Leu Asp Cys Asp HisTyr Val 450 455 460 Asn Asn Ser Lys Ala Val Arg Glu Ala Met Cys Phe LeuMet Asp Pro 465 470 475 480 Gln Val Gly Arg Asp Val Cys Tyr Val Gln PhePro Gln Arg Phe Asp 485 490 495 Gly Ile Asp Arg Ser Asp Arg Tyr Ala AsnArg Asn Thr Val Phe Phe 500 505 510 Asp Val Asn Met Lys Gly Leu Asp GlyIle Gln Gly Pro Val Tyr Val 515 520 525 Gly Thr Gly Cys Val Phe Asn ArgGln Ala Leu Tyr Gly Tyr Gly Pro 530 535 540 Pro Ser Met Pro Ser Phe ProLys Ser Ser Ser Ser Ser Cys Ser Cys 545 550 555 560 Cys Cys Pro Gly LysLys Glu Pro Lys Asp Pro Ser Glu Leu Tyr Arg 565 570 575 Asp Ala Lys ArgGlu Glu Leu Asp Ala Ala Ile Phe Asn Leu Arg Glu 580 585 590 Ile Asp AsnTyr Asp Glu Tyr Glu Arg Ser Met Leu Ile Ser Gln Thr 595 600 605 Ser PheGlu Lys Thr Phe Gly Leu Ser Ser Val Phe Ile Glu Ser Thr 610 615 620 LeuMet Glu Asn Gly Gly Val Ala Glu Ser Ala Asn Pro Ser Thr Leu 625 630 635640 Ile Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu Glu Lys Thr 645650 655 Ala Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp660 665 670 Ile Leu Thr Gly Phe Lys Met His Cys Arg Gly Trp Arg Ser IleTyr 675 680 685 Cys Met Pro Leu Arg Pro Ala Phe Lys Gly Ser Ala Pro IleAsn Leu 690 695 700 Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala Leu GlySer Val Glu 705 710 715 720 Ile Phe Leu Ser Arg His Cys Pro Leu Trp TyrGly Phe Gly Gly Gly 725 730 735 Arg Leu Lys Trp Leu Gln Arg Leu Ala TyrIle Asn Thr Ile Val Tyr 740 745 750 Pro Phe Thr Ser Leu Pro Leu Ile AlaTyr Cys Ser Leu Pro Ala Ile 755 760 765 Cys Leu Leu Thr Gly Lys Phe IleIle Pro Thr Leu Ser Asn Leu Ala 770 775 780 Ser Val Leu Phe Leu Gly LeuPhe Leu Ser Ile Ile Val Thr Ala Val 785 790 795 800 Leu Glu Leu Arg TrpSer Gly Val Ser Ile Glu Asp Leu Trp Arg Asn 805 810 815 Glu Gln Phe TrpVal Ile Gly Gly Val Ser Ala His Leu Phe Ala Val 820 825 830 Phe Gln GlyPhe Leu Lys Met Leu Ala Gly Ile Asp Thr Asn Phe Thr 835 840 845 Val ThrAla Lys Ala Ala Asp Asp Ala Asp Phe Gly Glu Leu Tyr Ile 850 855 860 ValLys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu Leu Ile Val 865 870 875880 Asn Met Val Gly Val Val Ala Gly Phe Ser Asp Ala Leu Asn Lys Gly 885890 895 Tyr Glu Ala Trp Gly Pro Leu Phe Gly Lys Val Phe Phe Ser Phe Trp900 905 910 Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly ArgGln 915 920 925 Asn Arg Thr Pro Thr Ile Val Val Leu Trp Ser Val Leu LeuAla Ser 930 935 940 Val Phe Ser Leu Val Trp Val Arg Ile Asn Pro Phe ValSer Thr Ala 945 950 955 960 Asp Ser Thr Thr Val Ser Gln Ser Cys Ile SerIle Asp Cys 965 970 7 685 PRT Gossypium hirsutum 7 Ala Arg Arg Trp ValPro Phe Cys Lys Lys His Asn Val Glu Pro Arg 1 5 10 15 Ala Pro Glu PheTyr Phe Asn Glu Lys Ile Asp Tyr Leu Lys Asp Lys 20 25 30 Val His Pro SerPhe Val Lys Glu Arg Arg Ala Met Lys Arg Glu Tyr 35 40 45 Glu Glu Phe LysVal Arg Ile Asn Ala Leu Val Ala Lys Ala Gln Lys 50 55 60 Lys Pro Glu GluGly Trp Val Met Gln Asp Gly Thr Pro Trp Pro Gly 65 70 75 80 Asn Asn ThrArg Asp His Pro Gly Met Ile Gln Val Tyr Leu Gly Ser 85 90 95 Ala Gly AlaLeu Asp Val Asp Gly Lys Glu Leu Pro Arg Leu Val Tyr 100 105 110 Val SerArg Glu Lys Arg Pro Gly Tyr Gln His His Lys Lys Ala Gly 115 120 125 AlaGlu Asn Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn Ala Pro 130 135 140Phe Ile Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala 145 150155 160 Met Arg Glu Ala Met Cys Phe Leu Met Asp Pro Gln Phe Gly Lys Lys165 170 175 Leu Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp ArgHis 180 185 190 Asp Arg Tyr Ala Asn Arg Asn Val Val Phe Phe Asp Ile AsnMet Leu 195 200 205 Gly Leu Asp Gly Leu Gln Gly Pro Val Tyr Val Gly ThrGly Cys Val 210 215 220 Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp Pro ProVal Ser Glu Lys 225 230 235 240 Arg Pro Lys Met Thr Cys Asp Cys Trp ProSer Trp Cys Cys Cys Cys 245 250 255 Cys Gly Gly Ser Arg Lys Lys Ser LysLys Lys Gly Glu Lys Lys Gly 260 265 270 Leu Leu Gly Gly Leu Leu Tyr GlyLys Lys Lys Lys Met Met Gly Lys 275 280 285 Asn Tyr Val Lys Lys Gly SerAla Pro Val Phe Asp Leu Glu Glu Ile 290 295 300 Glu Glu Gly Leu Glu GlyTyr Glu Glu Leu Glu Lys Ser Thr Leu Met 305 310 315 320 Ser Gln Lys AsnPhe Glu Lys Arg Phe Gly Gln Ser Pro Val Phe Ile 325 330 335 Ala Ser ThrLeu Met Glu Asn Gly Gly Leu Pro Glu Gly Thr Asn Ser 340 345 350 Thr SerLeu Ile Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu 355 360 365 GluLys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Val 370 375 380Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Cys Arg Gly Trp Lys 385 390395 400 Ser Val Tyr Cys Val Pro Lys Arg Pro Ala Phe Lys Gly Ser Ala Pro405 410 415 Ile Asn Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala LeuGly 420 425 430 Ser Val Glu Ile Phe Leu Ser Arg His Cys Pro Leu Trp TyrGly Tyr 435 440 445 Gly Gly Lys Leu Lys Trp Leu Glu Arg Leu Ala Tyr IleAsn Thr Ile 450 455 460 Val Tyr Pro Phe Thr Ser Ile Pro Leu Leu Ala TyrCys Thr Ile Pro 465 470 475 480 Ala Val Cys Leu Leu Thr Gly Lys Phe IleIle Pro Thr Leu Ser Asn 485 490 495 Leu Thr Ser Val Trp Phe Leu Ala LeuPhe Leu Ser Ile Ile Ala Thr 500 505 510 Gly Val Leu Glu Leu Arg Trp SerGly Val Ser Ile Gln Asp Trp Trp 515 520 525 Arg Asn Glu Gln Phe Trp ValIle Gly Gly Val Ser Ala His Leu Phe 530 535 540 Ala Val Phe Gln Gly LeuLeu Lys Val Leu Ala Gly Val Asp Thr Asn 545 550 555 560 Phe Thr Val ThrAla Lys Ala Ala Asp Asp Thr Glu Phe Gly Glu Leu 565 570 575 Tyr Leu PheLys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu Ile 580 585 590 Ile LeuAsn Met Val Gly Val Val Ala Gly Val Ser Asp Ala Ile Asn 595 600 605 AsnGly Tyr Gly Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala 610 615 620Phe Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly 625 630635 640 Arg Gln Asn Arg Thr Pro Thr Ile Val Val Leu Trp Ser Ile Leu Leu645 650 655 Ala Ser Ile Phe Ser Leu Val Trp Val Arg Ile Asp Pro Phe LeuPro 660 665 670 Lys Gln Thr Gly Pro Val Leu Lys Gln Cys Gly Val Glu 675680 685 8 881 PRT Oryzae sativa 8 Gly Asn Val Ala Trp Lys Glu Arg ValAsp Gly Trp Lys Leu Lys Gln 1 5 10 15 Asp Lys Gly Ala Ile Pro Met ThrAsn Gly Thr Ser Ile Ala Pro Ser 20 25 30 Glu Gly Arg Gly Val Gly Asp IleAsp Ala Ser Thr Asp Tyr Asn Asn 35 40 45 Glu Asp Ala Leu Leu Asn Asp GluThr Arg Gln Pro Leu Ser Arg Lys 50 55 60 Val Pro Leu Pro Ser Ser Arg IleAsn Pro Tyr Arg Asn Val Ile Val 65 70 75 80 Leu Arg Leu Val Val Leu SerIle Phe Leu His Tyr Arg Ile Thr Asn 85 90 95 Pro Val Arg Asn Ala Tyr ProLeu Trp Leu Leu Ser Val Ile Cys Glu 100 105 110 Ile Trp Phe Ala Leu SerTrp Leu Ile Asp Gln Phe Pro Lys Trp Phe 115 120 125 Pro Ile Asn Arg GluThr Tyr Leu Asp Arg Leu Ala Leu Arg Tyr Asp 130 135 140 Arg Glu Gly GluPro Ser Gln Leu Ala Ala Val Asp Ile Phe Val Ser 145 150 155 160 Thr ValAsp Pro Met Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val 165 170 175 LeuSer Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr 180 185 190Val Ser Asp Asp Gly Ala Ala Met Leu Thr Phe Asp Ala Leu Ala Glu 195 200205 Thr Ser Glu Phe Ala Arg Lys Trp Val Pro Phe Val Lys Lys Tyr Asn 210215 220 Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe Ser Gln Lys Ile Asp Tyr225 230 235 240 Leu Lys Asp Lys Val His Pro Ser Phe Val Lys Asp Arg ArgAla Met 245 250 255 Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn GlyLeu Val Ala 260 265 270 Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Ile MetGln Asp Gly Thr 275 280 285 Pro Trp Pro Gly Asn Asn Thr Arg Asp His ProGly Met Ile Gln Val 290 295 300 Phe Leu Gly His Ser Gly Gly Leu Asp ThrGlu Gly Asn Glu Leu Pro 305 310 315 320 Arg Leu Val Tyr Val Ser Arg GluLys Arg Pro Gly Phe Gln His His 325 330 335 Lys Lys Ala Gly Ala Met AsnAla Leu Val Arg Val Ser Ala Val Leu 340 345 350 Thr Asn Gly Gln Tyr MetLeu Asn Leu Asp Cys Asp His Tyr Ile Asn 355 360 365 Asn Ser Lys Ala LeuArg Glu Ala Met Cys Phe Leu Met Asp Pro Asn 370 375 380 Leu Gly Arg SerVal Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly 385 390 395 400 Ile AspArg Asn Asp Arg Tyr Ala Asn Arg Asn Thr Val Phe Phe Asp 405 410 415 IleAsn Leu Arg Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly 420 425 430Thr Gly Cys Val Phe Asn Arg Thr Ala Leu Tyr Gly Tyr Glu Pro Pro 435 440445 Ile Lys Gln Lys Lys Lys Gly Ser Phe Leu Ser Ser Leu Cys Gly Gly 450455 460 Arg Lys Lys Ala Ser Lys Ser Lys Lys Lys Ser Ser Asp Lys Lys Lys465 470 475 480 Ser Asn Lys His Val Asp Ser Ala Val Pro Val Phe Asn LeuGlu Asp 485 490 495 Ile Glu Glu Gly Val Glu Gly Ala Gly Phe Asp Asp GluLys Ser Leu 500 505 510 Leu Met Ser Gln Met Ser Leu Glu Lys Arg Phe GlyGln Ser Ala Ala 515 520 525 Phe Val Ala Ser Thr Leu Met Glu Tyr Gly GlyVal Pro Gln Ser Ala 530 535 540 Thr Pro Glu Ser Leu Leu Lys Glu Ala IleHis Val Ile Ser Cys Gly 545 550 555 560 Tyr Glu Asp Lys Thr Glu Trp GlyThr Glu Ile Gly Trp Ile Tyr Gly 565 570 575 Ser Val Thr Glu Asp Ile LeuThr Gly Phe Lys Met His Ala Arg Gly 580 585 590 Trp Arg Ser Ile Tyr CysMet Pro Lys Arg Pro Ala Phe Lys Gly Ser 595 600 605 Ala Pro Ile Asn LeuSer Asp Arg Leu Asn Gln Val Leu Arg Trp Ala 610 615 620 Leu Gly Ser ValGlu Ile Leu Phe Ser Arg His Cys Pro Ile Trp Tyr 625 630 635 640 Gly TyrGly Gly Arg Leu Lys Phe Leu Glu Arg Phe Ala Tyr Ile Asn 645 650 655 ThrThr Ile Tyr Pro Leu Thr Ser Ile Pro Leu Leu Ile Tyr Cys Val 660 665 670Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Glu Ile 675 680685 Ser Asn Phe Ala Ser Ile Trp Phe Ile Ser Leu Phe Ile Ser Ile Phe 690695 700 Ala Thr Gly Ile Leu Glu Met Arg Trp Ser Gly Val Gly Ile Asp Glu705 710 715 720 Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Ile SerAla His 725 730 735 Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu AlaGly Ile Asp 740 745 750 Thr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp GluAsp Gly Asp Phe 755 760 765 Ala Glu Leu Tyr Met Phe Lys Trp Thr Thr LeuLeu Ile Pro Pro Thr 770 775 780 Thr Ile Leu Ile Ile Asn Leu Val Gly ValVal Ala Gly Ile Ser Tyr 785 790 795 800 Ala Ile Asn Ser Gly Tyr Gln SerTrp Gly Pro Leu Phe Gly Lys Leu 805 810 815 Phe Phe Ala Phe Trp Val IleVal His Leu Tyr Pro Phe Leu Lys Gly 820 825 830 Leu Met Gly Arg Gln AsnArg Thr Pro Thr Ile Val Val Val Trp Ala 835 840 845 Ile Leu Leu Ala SerIle Phe Ser Leu Leu Trp Val Arg Ile Asp Pro 850 855 860 Phe Thr Thr ArgVal Thr Gly Pro Asp Thr Gln Thr Cys Gly Ile Asn 865 870 875 880 Cys 9723 PRT Acetobacter xylinum 9 Met Pro Glu Val Arg Ser Ser Thr Gln SerGlu Ser Gly Met Ser Gln 1 5 10 15 Trp Met Gly Lys Ile Leu Ser Ile ArgGly Ala Gly Leu Thr Ile Gly 20 25 30 Val Phe Gly Leu Cys Ala Leu Ile AlaAla Thr Ser Val Thr Leu Pro 35 40 45 Pro Glu Gln Gln Leu Ile Val Ala PheVal Cys Val Val Ile Phe Phe 50 55 60 Ile Val Gly His Lys Pro Ser Arg ArgSer Gln Ile Phe Leu Glu Val 65 70 75 80 Leu Ser Gly Leu Val Ser Leu ArgTyr Leu Thr Trp Arg Leu Thr Glu 85 90 95 Thr Leu Ser Phe Asp Thr Trp LeuGln Gly Leu Leu Gly Thr Met Leu 100 105 110 Leu Val Ala Glu Leu Tyr AlaLeu Met Met Leu Phe Leu Ser Tyr Phe 115 120 125 Gln Thr Ile Ala Pro LeuHis Arg Ala Pro Leu Pro Leu Pro Pro Asn 130 135 140 Pro Asp Glu Trp ProThr Val Asp Ile Phe Val Pro Thr Tyr Asn Glu 145 150 155 160 Glu Leu SerIle Val Arg Leu Thr Val Leu Gly Ser Leu Gly Ile Asp 165 170 175 Trp ProPro Glu Lys Val Arg Val His Ile Leu Asp Asp Gly Arg Arg 180 185 190 ProGlu Phe Ala Ala Phe Ala Ala Glu Cys Gly Ala Asn Tyr Ile Ala 195 200 205Arg Pro Thr Asn Glu His Ala Lys Ala Gly Asn Leu Asn Tyr Ala Ile 210 215220 Gly His Thr Asp Gly Asp Tyr Ile Leu Ile Phe Asp Cys Asp His Val 225230 235 240 Pro Thr Arg Ala Phe Leu Gln Leu Thr Met Gly Trp Met Val GluAsp 245 250 255 Pro Lys Ile Ala Leu Met Gln Thr Pro His His Phe Tyr SerPro Asp 260 265 270 Pro Phe Gln Arg Asn Leu Ser Ala Gly Tyr Arg Thr ProPro Glu Gly 275 280 285 Asn Leu Phe Tyr Gly Val Val Gln Asp Gly Asn AspPhe Trp Asp Ala 290 295 300 Thr Phe Phe Cys Gly Ser Cys Ala Ile Leu ArgArg Thr Ala Ile Glu 305 310 315 320 Gln Ile Gly Gly Phe Ala Thr Gln ThrVal Thr Glu Asp Ala His Thr 325 330 335 Ala Leu Lys Met Gln Arg Leu GlyTrp Ser Thr Ala Tyr Leu Arg Ile 340 345 350 Pro Leu Ala Gly Gly Leu AlaThr Glu Arg Leu Ile Leu His Ile Gly 355 360 365 Gln Arg Val Arg Trp AlaArg Gly Met Leu Gln Ile Phe Arg Ile Asp 370 375 380 Asn Pro Leu Phe GlyArg Gly Leu Ser Trp Gly Gln Arg Leu Cys Tyr 385 390 395 400 Leu Ser AlaMet Thr Ser Phe Leu Phe Ala Val Pro Arg Val Ile Phe 405 410 415 Leu SerSer Pro Leu Ala Phe Leu Phe Phe Gly Gln Asn Ile Ile Ala 420 425 430 AlaSer Pro Leu Ala Leu Leu Ala Tyr Ala Ile Pro His Met Phe His 435 440 445Ala Val Gly Thr Ala Ser Lys Ile Asn Lys Gly Trp Arg Tyr Ser Phe 450 455460 Trp Ser Glu Val Tyr Glu Thr Thr Met Ala Leu Phe Leu Val Arg Val 465470 475 480 Thr Ile Val Thr Leu Leu Ser Pro Ser Arg Gly Lys Phe Asn ValThr 485 490 495 Asp Lys Gly Gly Leu Leu Glu Lys Gly Tyr Phe Asp Leu GlyAla Val 500 505 510 Tyr Pro Asn Ile Ile Leu Gly Leu Ile Met Phe Gly GlyLeu Ala Arg 515 520 525 Gly Val Tyr Glu Leu Ser Phe Gly His Leu Asp GlnIle Ala Glu Arg 530 535 540 Ala Tyr Leu Leu Asn Ser Ala Trp Ala Met LeuSer Leu Ile Ile Ile 545 550 555 560 Leu Ala Ala Ile Ala Val Gly Arg GluThr Gln Gln Lys Arg Asn Ser 565 570 575 His Arg Ile Pro Ala Thr Ile ProVal Glu Val Ala Asn Ala Asp Gly 580 585 590 Ser Ile Ile Val Thr Gly ValThr Glu Asp Leu Ser Met Gly Gly Ala 595 600 605 Ala Val Lys Met Ser TrpPro Ala Lys Leu Ser Gly Pro Thr Pro Val 610 615 620 Tyr Ile Arg Thr ValLeu Asp Gly Glu Glu Leu Ile Leu Pro Ala Arg 625 630 635 640 Ile Ile ArgAla Gly Asn Gly Arg Gly Ile Phe Ile Trp Thr Ile Asp 645 650 655 Asn LeuGln Gln Glu Phe Ser Val Ile Arg Leu Val Phe Gly Arg Ala 660 665 670 AspAla Trp Val Asp Leu Gly Gln Leu Gln Gly Arg Pro Pro Ala Ala 675 680 685Gln Pro His Gly His Gly Ser Gln Arg Gln Gly Pro Val Pro Phe Lys 690 695700 Trp Arg Tyr Arg Pro Ser Gln Phe Pro Asn Gln Ala Phe Gly Trp Gln 705710 715 720 Cys Pro Val 10 756 PRT acetobacter xylinum 10 Met Ser GluVal Gln Ser Pro Val Pro Thr Glu Ser Arg Leu Gly Arg 1 5 10 15 Ile SerAsn Lys Ile Leu Ser Leu Arg Gly Ala Ser Tyr Ile Val Gly 20 25 30 Ala LeuGly Leu Cys Ala Leu Ile Ala Ala Thr Thr Val Thr Leu Asn 35 40 45 Asn AsnGlu Gln Leu Ile Val Ala Ala Val Cys Val Val Ile Phe Phe 50 55 60 Val ValGly Arg Gly Lys Ser Arg Arg Thr Gln Ile Phe Leu Glu Val 65 70 75 80 LeuSer Ala Leu Val Ser Leu Arg Tyr Leu Thr Trp Arg Leu Thr Glu 85 90 95 ThrLeu Asp Phe Asn Thr Trp Ile Gln Gly Ile Leu Gly Val Ile Leu 100 105 110Leu Met Ala Glu Leu Tyr Ala Leu Tyr Met Leu Phe Leu Ser Tyr Phe 115 120125 Gln Thr Ile Gln Pro Leu His Arg Ala Pro Leu Pro Leu Pro Asp Asn 130135 140 Val Asp Asp Trp Pro Thr Val Asp Ile Phe Ile Pro Thr Tyr Asp Glu145 150 155 160 Gln Leu Ser Ile Val Arg Leu Thr Val Leu Gly Ala Leu GlyIle Asp 165 170 175 Trp Pro Pro Asp Lys Val Asn Val Tyr Ile Leu Asp AspGly Val Arg 180 185 190 Pro Glu Phe Glu Gln Phe Ala Lys Asp Cys Gly AlaLeu Tyr Ile Gly 195 200 205 Arg Val Asp Val Asp Ser Ala His Ala Lys AlaGly Asn Leu Asn His 210 215 220 Ala Ile Lys Arg Thr Ser Gly Asp Tyr IleLeu Ile Leu Asp Cys Asp 225 230 235 240 His Ile Pro Thr Arg Ala Phe LeuGln Ile Ala Met Gly Trp Met Val 245 250 255 Ala Asp Arg Lys Ile Ala LeuMet Gln Thr Pro His His Phe Tyr Ser 260 265 270 Pro Asp Pro Phe Gln ArgAsn Leu Ala Val Gly Tyr Arg Thr Pro Pro 275 280 285 Glu Gly Asn Leu PheTyr Gly Val Ile Gln Asp Gly Asn Asp Phe Trp 290 295 300 Asp Ala Thr PhePhe Cys Gly Ser Cys Ala Ile Leu Arg Arg Glu Ala 305 310 315 320 Ile GluSer Ile Gly Gly Phe Ala Val Glu Thr Val Thr Glu Asp Ala 325 330 335 HisThr Ala Leu Arg Met Gln Arg Arg Gly Trp Ser Thr Ala Tyr Leu 340 345 350Arg Ile Pro Val Ala Ser Gly Leu Ala Thr Glu Arg Leu Thr Thr His 355 360365 Ile Gly Gln Arg Met Arg Trp Ala Arg Gly Met Ile Gln Ile Phe Arg 370375 380 Val Asp Asn Pro Met Leu Gly Arg Gly Leu Lys Leu Gly Gln Arg Leu385 390 395 400 Cys Tyr Leu Ser Ala Met Thr Ser Phe Phe Phe Ala Ile ProArg Val 405 410 415 Ile Phe Leu Ala Ser Pro Leu Ala Phe Leu Phe Ala GlyGln Asn Ile 420 425 430 Ile Ala Ala Ala Pro Leu Ala Val Ala Ala Tyr AlaLeu Pro His Met 435 440 445 Phe His Ser Ile Ala Thr Ala Ala Lys Val AsnLys Gly Trp Arg Tyr 450 455 460 Ser Phe Trp Ser Glu Val Tyr Glu Thr ThrMet Ala Leu Phe Leu Val 465 470 475 480 Arg Val Thr Ile Val Thr Leu LeuPhe Pro Ser Lys Gly Lys Phe Asn 485 490 495 Val Thr Glu Lys Gly Gly ValLeu Glu Glu Glu Glu Phe Asp Leu Gly 500 505 510 Ala Thr Tyr Pro Asn IleIle Phe Ala Thr Ile Met Met Gly Gly Leu 515 520 525 Leu Ile Gly Leu PheGlu Leu Ile Val Arg Phe Asn Gln Leu Asp Val 530 535 540 Ile Ala Arg AsnAla Tyr Leu Leu Asn Cys Ala Trp Ala Leu Ile Ser 545 550 555 560 Leu IleIle Leu Phe Ala Ala Ile Ala Val Gly Arg Glu Thr Lys Gln 565 570 575 ValArg Tyr Asn His Arg Val Glu Ala His Ile Pro Val Thr Val Tyr 580 585 590Asp Ala Pro Ala Glu Gly Gln Pro His Thr Tyr Tyr Asn Ala Thr His 595 600605 Gly Met Thr Gln Asp Val Ser Met Gly Gly Val Ala Val His Ile Pro 610615 620 Leu Pro Asp Val Thr Thr Gly Pro Val Lys Lys Arg Ile His Ala Val625 630 635 640 Leu Asp Gly Glu Glu Ile Asp Ile Pro Ala Thr Met Leu ArgCys Thr 645 650 655 Asn Gly Lys Ala Val Phe Thr Trp Asp Asn Asn Asp LeuAsp Thr Glu 660 665 670 Arg Asp Ile Val Arg Phe Val Phe Gly Arg Ala AspAla Trp Leu Gln 675 680 685 Trp Asn Asn Tyr Glu Asp Asp Arg Pro Leu ArgSer Leu Trp Ser Leu 690 695 700 Leu Leu Ser Ile Lys Ala Leu Phe Arg LysLys Gly Lys Ile Met Ala 705 710 715 720 Asn Ser Arg Pro Lys Lys Lys ProLeu Ala Leu Pro Val Glu Arg Arg 725 730 735 Glu Pro Thr Thr Ile His SerGly Gln Thr Gln Glu Gly Lys Ile Ser 740 745 750 Arg Ala Ala Ser 755 11693 PRT Escherichia coli 11 Met Leu Leu Trp Gly Val Ala Leu Ile Val ArgArg Met Pro Gly Arg 1 5 10 15 Phe Ser Ala Leu Met Leu Ile Val Leu SerLeu Thr Val Ser Cys Arg 20 25 30 Tyr Ile Trp Trp Arg Tyr Thr Ser Thr LeuAsn Trp Asp Asp Pro Val 35 40 45 Ser Leu Val Cys Gly Leu Ile Leu Leu PheAla Ile Thr Tyr Ala Trp 50 55 60 Ile Val Leu Val Leu Gly Tyr Phe Gln ValVal Trp Pro Leu Asn Arg 65 70 75 80 Gln Pro Val Pro Leu Pro Lys Asp MetSer Leu Trp Pro Ser Val Asp 85 90 95 Ile Phe Val Pro Thr Tyr Asn Glu AspLeu Asn Val Val Lys Asn Thr 100 105 110 Ile Tyr Ala Ser Leu Gly Ile AspTrp Pro Lys Asp Lys Leu Asn Ile 115 120 125 Trp Ile Leu Asp Asp Gly GlyArg Glu Glu Phe Arg Gln Phe Ala Gln 130 135 140 Asn Val Gly Val Lys TyrIle Ala Arg Thr Thr His Glu His Ala Lys 145 150 155 160 Ala Gly Asn IleAsn Asn Ala Leu Lys Tyr Ala Lys Gly Glu Phe Val 165 170 175 Ser Ile PheAsp Cys Asp His Val Pro Thr Arg Ser Phe Leu Gln Met 180 185 190 Thr MetGly Trp Phe Leu Lys Glu Lys Gln Leu Ala Met Met Gln Thr 195 200 205 ProHis His Phe Phe Ser Pro Asp Pro Phe Glu Arg Asn Leu Gly Arg 210 215 220Phe Arg Lys Thr Pro Asn Glu Gly Thr Leu Phe Tyr Gly Leu Val Gln 225 230235 240 Asp Gly Asn Asp Met Trp Asp Ala Thr Phe Phe Cys Gly Ser Cys Ala245 250 255 Val Ile Arg Arg Lys Pro Leu Asp Glu Ile Gly Gly Ile Ala ValGlu 260 265 270 Thr Val Thr Glu Asp Ala His Thr Ser Leu Arg Leu His ArgArg Gly 275 280 285 Tyr Thr Ser Ala Tyr Met Arg Ile Pro Gln Ala Ala GlyLeu Ala Thr 290 295 300 Glu Ser Leu Ser Ala His Ile Gly Gln Arg Ile ArgTrp Ala Arg Gly 305 310 315 320 Met Val Gln Ile Phe Arg Leu Asp Asn ProLeu Thr Gly Lys Gly Leu 325 330 335 Lys Phe Ala Gln Arg Leu Cys Tyr ValAsn Ala Met Phe His Phe Leu 340 345 350 Ser Gly Ile Pro Arg Leu Ile PheLeu Thr Ala Pro Leu Ala Phe Leu 355 360 365 Leu Leu His Ala Tyr Ile IleTyr Ala Pro Ala Leu Met Ile Ala Leu 370 375 380 Phe Val Leu Pro His MetIle His Ala Ser Leu Thr Asn Ser Lys Ile 385 390 395 400 Gln Gly Lys TyrArg His Ser Phe Trp Ser Glu Ile Tyr Glu Thr Val 405 410 415 Leu Ala TrpTyr Ile Ala Pro Pro Thr Leu Val Ala Leu Ile Asn Pro 420 425 430 His LysGly Lys Phe Asn Val Thr Ala Lys Gly Gly Gly Leu Val Glu 435 440 445 GluGlu Tyr Val Asp Trp Val Ile Ser Arg Pro Tyr Ile Phe Leu Val 450 455 460Leu Leu Asn Leu Val Gly Val Ala Val Gly Ile Trp Arg Tyr Phe Tyr 465 470475 480 Gly Pro Pro Thr Glu Met Leu Thr Val Val Val Ser Met Val Trp Val485 490 495 Phe Tyr Asn Leu Ile Val Leu Gly Gly Ala Val Ala Val Ser ValGlu 500 505 510 Ser Lys Gln Val Arg Arg Ser His Arg Val Glu Met Thr MetPro Ala 515 520 525 Ala Ile Ala Arg Glu Asp Gly His Leu Phe Ser Cys ThrVal Gln Asp 530 535 540 Phe Ser Asp Gly Gly Leu Gly Ile Lys Ile Asn GlyGln Ala Gln Ile 545 550 555 560 Leu Glu Gly Gln Lys Val Asn Leu Leu LeuLys Arg Gly Gln Gln Glu 565 570 575 Tyr Val Phe Pro Thr Gln Val Ala ArgVal Met Gly Asn Glu Val Gly 580 585 590 Leu Lys Leu Met Pro Leu Thr ThrGln Gln His Ile Asp Phe Val Gln 595 600 605 Cys Thr Phe Ala Arg Ala AspThr Trp Ala Leu Trp Gln Asp Ser Tyr 610 615 620 Pro Glu Asp Lys Pro LeuGlu Ser Leu Leu Asp Ile Leu Lys Leu Gly 625 630 635 640 Phe Arg Gly TyrArg His Leu Ala Glu Phe Ala Pro Ser Ser Val Lys 645 650 655 Gly Ile PheArg Val Leu Thr Ser Leu Val Ser Trp Val Val Ser Phe 660 665 670 Ile ProPro Arg Pro Glu Arg Ser Glu Thr Ala Gln Pro Ser Asp Gln 675 680 685 AlaLeu Ala Gln Gln 690 12 861 PRT Agrobacterium tumefaciens 12 Met Cys ArgCys Gly Arg Ala Val Arg Ser Arg Pro Val Cys Arg Pro 1 5 10 15 Gly GlnLeu Val Val Arg Arg Ser Pro Arg Pro Arg Ser Arg Asn His 20 25 30 Ser ArgCys Arg Pro Leu Arg Leu Ser Val Phe Pro Arg Pro His Arg 35 40 45 Arg ValArg His His Cys Gln Arg Asp Leu Arg Trp Glu Pro Gly Arg 50 55 60 Trp IleAla Val Arg Trp Lys Ala Ala Arg Ser His Arg Arg Phe Arg 65 70 75 80 ArgCys Pro Phe Pro Arg Gln Leu Val Trp Pro Val Arg Glu Arg His 85 90 95 ArgAsp Ala Gly Asp Arg Arg Asn Gln Arg Glu Arg Arg Arg Arg Asp 100 105 110Ala Tyr His Glu Ile Ser Glu Pro Lys Phe Arg Thr Arg Lys Arg Thr 115 120125 Glu Ser Phe Trp Met Asn Lys Ala Ile Thr Val Ile Val Trp Leu Leu 130135 140 Val Ser Leu Cys Val Leu Ala Ile Ile Thr Met Pro Val Ser Leu Gln145 150 155 160 Thr His Leu Val Ala Thr Ala Ile Ser Leu Ile Leu Leu AlaThr Ile 165 170 175 Lys Ser Phe Asn Gly Gln Gly Ala Trp Arg Leu Val AlaLeu Gly Phe 180 185 190 Gly Thr Ala Ile Val Leu Arg Tyr Val Tyr Trp ArgThr Thr Ser Thr 195 200 205 Leu Pro Pro Val Asn Gln Leu Glu Asn Phe IlePro Gly Phe Leu Leu 210 215 220 Tyr Leu Ala Glu Met Tyr Ser Val Val MetLeu Gly Leu Ser Leu Val 225 230 235 240 Ile Val Ser Met Pro Leu Pro SerArg Lys Thr Arg Pro Gly Ser Pro 245 250 255 Asp Tyr Arg Pro Thr Val AspVal Phe Val Pro Ser Tyr Asn Glu Asp 260 265 270 Ala Glu Leu Leu Ala AsnThr Leu Ala Ala Ala Lys Asn Met Asp Tyr 275 280 285 Pro Ala Asp Arg PheThr Val Trp Leu Leu Asp Asp Gly Gly Ser Val 290 295 300 Gln Lys Arg AsnAla Ala Asn Ile Val Glu Ala Gln Ala Ala Gln Arg 305 310 315 320 Arg HisGlu Glu Leu Lys Lys Leu Cys Glu Asp Leu Asp Val Arg Tyr 325 330 335 LeuThr Arg Glu Arg Asn Val His Ala Lys Ala Gly Asn Leu Asn Asn 340 345 350Gly Leu Ala His Ser Thr Gly Glu Leu Val Thr Val Phe Asp Ala Asp 355 360365 His Ala Pro Ala Arg Asp Phe Leu Leu Glu Thr Val Gly Tyr Phe Asp 370375 380 Glu Asp Pro Arg Leu Phe Leu Val Gln Thr Pro His Phe Phe Val Asn385 390 395 400 Pro Asp Pro Ile Glu Arg Asn Leu Arg Thr Phe Glu Thr MetPro Ser 405 410 415 Glu Asn Glu Met Phe Tyr Gly Ile Ile Gln Arg Gly LeuAsp Lys Trp 420 425 430 Asn Gly Ala Phe Phe Cys Gly Ser Ala Ala Val LeuArg Arg Glu Ala 435 440 445 Leu Gln Asp Ser Asp Gly Phe Ser Gly Val SerIle Thr Glu Asp Cys 450 455 460 Glu Thr Ala Leu Ala Leu His Ser Arg GlyTrp Asn Ser Val Tyr Val 465 470 475 480 Asp Lys Pro Leu Ile Ala Gly LeuGln Pro Ala Thr Phe Ala Ser Phe 485 490 495 Ile Gly Gln Arg Ser Arg TrpAla Gln Gly Met Met Gln Ile Leu Ile 500 505 510 Phe Arg Gln Pro Leu PheLys Arg Gly Leu Ser Phe Thr Gln Arg Leu 515 520 525 Cys Tyr Met Ser SerThr Leu Phe Trp Leu Phe Pro Phe Pro Arg Thr 530 535 540 Ile Phe Leu PheAla Pro Leu Phe Tyr Leu Phe Phe Asp Leu Gln Ile 545 550 555 560 Phe ValAla Ser Gly Gly Glu Phe Leu Ala Tyr Thr Ala Ala Tyr Met 565 570 575 LeuVal Asn Leu Met Met Gln Asn Tyr Leu Tyr Gly Ser Phe Arg Trp 580 585 590Pro Trp Ile Ser Glu Leu Tyr Glu Tyr Val Gln Thr Val His Leu Leu 595 600605 Pro Ala Val Val Ser Val Ile Phe Asn Pro Gly Lys Pro Thr Phe Lys 610615 620 Val Thr Ala Lys Asp Glu Ser Ile Ala Glu Ala Arg Leu Ser Glu Ile625 630 635 640 Ser Arg Pro Phe Phe Val Ile Phe Ala Leu Leu Leu Val AlaMet Ala 645 650 655 Phe Ala Val Trp Arg Ile Tyr Ser Glu Pro Tyr Lys AlaAsp Val Thr 660 665 670 Leu Val Val Gly Gly Trp Asn Leu Leu Asn Leu IlePhe Ala Gly Cys 675 680 685 Ala Leu Gly Val Val Ser Glu Arg Gly Asp LysSer Ala Ser Arg Arg 690 695 700 Ile Thr Val Lys Arg Arg Cys Glu Val GlnLeu Gly Gly Ser Asp Thr 705 710 715 720 Trp Val Pro Ala Ser Ile Asp AsnVal Ser Val His Gly Leu Leu Ile 725 730 735 Asn Ile Phe Asp Ser Ala ThrAsn Ile Glu Lys Gly Ala Thr Ala Ile 740 745 750 Val Lys Val Lys Pro HisSer Glu Gly Val Pro Glu Thr Met Pro Leu 755 760 765 Asn Val Val Arg ThrVal Arg Gly Glu Gly Phe Val Ser Ile Gly Cys 770 775 780 Thr Phe Ser ProGln Arg Ala Val Asp His Arg Leu Ile Ala Asp Leu 785 790 795 800 Ile PheAla Asn Ser Glu Gln Trp Ser Glu Phe Gln Arg Val Arg Arg 805 810 815 LysLys Pro Gly Leu Ile Arg Gly Thr Ala Ile Phe Leu Ala Ile Ala 820 825 830Leu Phe Gln Thr Gln Arg Gly Leu Tyr Tyr Leu Val Arg Ala Arg Arg 835 840845 Pro Ala Pro Lys Ser Ala Lys Pro Val Gly Ala Val Lys 850 855 860

What is claimed is:
 1. An isolated polynucleotide comprising nucleotides1 through 953 of SEQ ID NO:3.
 2. A recombinant nucleic acid constructcomprising the isolated polynucleotide according to claim
 1. 3. Theconstruct according to claim 2 further comprising a secondpolynucleotide sequence of interest.
 4. A plant cell comprising theconstruct according to claim
 2. 5. A plant comprising the cell accordingto claim
 4. 6. A method of modifying fiber phenotype in a cotton plant,said method comprising: transforming a plant cell with the constructaccording to claim 2, and regenerating said plant cell to produce acotton plant with a modified fiber phenotype compared to anon-transformed cotton plant.